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MVAPACK Online

2024-11-08T02:06:05Z

Mjeppesen:

[[category:MVAPACK]]
==Introduction==

MVAPACK Online

2024-11-08T01:27:02Z

Mjeppesen:

[[category:MVAPACK]]

MVAPACK Online

2024-11-08T01:26:21Z

Mjeppesen: Created page with "[category:MVAPACK]"

[category:MVAPACK]

Tube deep cleaning

2024-09-09T15:35:11Z

Mjeppesen:

[[Category:General_Maintenance]]
[[Category:Metabolomics]]

1. Empty all contents from dirty NMR tubes into appropriate residue container. Remove the labels on the NMR tube by rinsing with ethanol.

2. Fill the container outside the protect bottles with liquid nitrogen and cool it down.

3. Load the tubes into the tube cleaning apparatus (tube washer) with proper solvents as noted below.

4. The operation of the tube washer:

a)With the left valve closed, slowly and briefly opening the T-valve to the vacuum position evacuates the air from the Labconco®
vacuum bottle and places it under a slight vacuum without removing the solvent.
b)Turn the T-valve to the vent position opens the vacuum bottle to atmosphere breaking the vacuum and forcing solvent into the NMR tube.
c)Switch the T-valve back to the vacuum position places the NMR tubes back under vacuum and remove the solvent from the NMR tube.
d)Repeat the process two or three times effectively washes the NMR tubes with solvent.
e)Switch the T-valve to the vent position and opening the left valve rapidly removes the solvent from the NMR tube cleaner into the
filter flask. The process can be repeated with other solvents.

5. Rinse NMR tubes with nano pure water by filling with water then empty all contents from dirty NMR tubes into appropriate residue container.

6. Rinse NMR tubes with ethanol by filling with ethanol then empty all contents from dirty NMR tubes into appropriate residue container.

7. Rinse NMR tubes with acetone by filling with acetone then empty all contents from dirty NMR tubes into appropriate residue container. The amount of organic solvents should be well-controlled.

8. Fill tubes with nano pure water.

9. Place NMR tubes in a concentrated nitric acid bath and soak 24hrs.

10. Empty contents of NMR tubes into the acid bath.

11. Rinse the NMR tubes under the tap water to remove most of the acid.

12. Load the tubes back to the tube washer.

13. Rinse NMR tubes in baking soda solution to neutralize acid.

14. Rinse NMR tube thoroughly with nano pure water until the solvent waste is neutral.

15. Dry NMR tubes: lay tubes horizontally in the oven.

16. Once dry inspect tubes for any crack, divots or other defect. The tubes with crack on the opening should be collected for further modification and reuse. Other problematic tubes have to be disposed.

[[File:Tubewasher1.jpg]][[File:Tubewasher2.jpg]]

A demonstration video can be found at here [https://bionmr.unl.edu/files/misc/tubewasher-instructions.wmv]

Processing CEST Data

2024-09-09T15:30:56Z

Mjeppesen:

Processing CEST Data

# Have X11, nmrPipe, Python >=2.7, and ChemEx (from the Kay lab) installed
# Open a c-shell with “csh” and navigate to the directory with your ser file
# Open X11
# In the terminal type “bruker”
# Click “read parameters”
# Switch the F1 and F2 axes
##y should be real with N total and valid points
##z should be the 15N dimension
## zMODE Echo-Antiecho
## yMODE Real
## aq2D States
# Save the script. It should look like the script that follows this protocol.
# Add these lines:
##xyz2pipe –in fid/test%03d.fid –z –ri2c
##| pipe2xyz –out fids/UBQ%03d.fid –y –ov
##/bin/rm –fr data/
# Run the [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/fid.com fid.com] script you just saved.
# Input file names and phase values into [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/ftcpmg.com ftcpmg.com] (follows this protocol) and run
# Open test spectrum in nmrDraw
# Pick peaks and save the peak list
# Rename the peaks to their corresponding amino acid and residue #. Alternatively, create a peakList.txt with their IDs and run [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/addA.py addA.py].
# Run [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/autoFit.tcl autoFit.tcl] in the c-shell.
##autoFit.tcl –specName fts/UBQ%03d.ft2 –inTab test.tab –series
# Run [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/extract_profiles_bru.py extract_profiles_bru.py] (follows)
##./extract_profiles_bru.py –tbl nlin.tab –par fq3list –out fit/
# Make sure the .out files are named like “A#N-HN.out”
# Then run ChemEx, check their tutorial

Other Helpful Files: [http://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/calcFMFinput.py calcFMFinput.py] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/exp.cfg exp.cfg] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/fit.com fit.com] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/met.cfg met.cfg] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/par.cfg par.cfg]

[[category:Needs_Updating]] 
[[category:Data_Processing_and_Analysis]]

Processing CEST Data

2024-09-09T15:25:18Z

Mjeppesen: Updated Links

Processing CEST Data

# Have X11, nmrPipe, Python >=2.7, and ChemEx (from the Kay lab) installed
# Open a c-shell with “csh” and navigate to the directory with your ser file
# Open X11
# In the terminal type “bruker”
# Click “read parameters”
# Switch the F1 and F2 axes
##y should be real with N total and valid points
##z should be the 15N dimension
## zMODE Echo-Antiecho
## yMODE Real
## aq2D States
# Save the script. It should look like the script that follows this protocol.
# Add these lines:
##xyz2pipe –in fid/test%03d.fid –z –ri2c
##| pipe2xyz –out fids/UBQ%03d.fid –y –ov
##/bin/rm –fr data/
# Run the [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/fid.com fid.com] script you just saved.
# Input file names and phase values into [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/ftcpmg.com ftcpmg.com] (follows this protocol) and run
# Open test spectrum in nmrDraw
# Pick peaks and save the peak list
# Rename the peaks to their corresponding amino acid and residue #. Alternatively, create a peakList.txt with their IDs and run [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/caddA.py addA.py].
# Run [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/autoFit.tcl autoFit.tcl] in the c-shell.
##autoFit.tcl –specName fts/UBQ%03d.ft2 –inTab test.tab –series
# Run [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/extract_profiles_bru.py extract_profiles_bru.py] (follows)
##./extract_profiles_bru.py –tbl nlin.tab –par fq3list –out fit/
# Make sure the .out files are named like “A#N-HN.out”
# Then run ChemEx, check their tutorial

Other Helpful Files: [http://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/calcFMFinput.py calcFMFinput.py] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/exp.cfg exp.cfg] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/fit.com fit.com] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/met.cfg met.cfg] [https://bionmr.unl.edu/files/misc/mediawikidownloads/CEST/par.cfg par.cfg]

[[category:Needs_Updating]] 
[[category:Data_Processing_and_Analysis]]

Example Scripts

2024-08-12T23:34:16Z

Mjeppesen: /* Example 1: 1D NMR Processing to Matrix Export */

=Example Scripts for Various MVAPACK Patterns=
''Note: Patterns may be sensitive to file location and path variables.''

===Example 1: 1D NMR Processing to Matrix Export===
'''''%%''''' indicates comment line. All lines can be copied and pasted to terminal.

<code>
%% Start file extraction from 1D NMR files located in nmrdata sub directory 
filenames = glob("nmrdata/*"); 
[F.data, F.parms] = loadnmr(filenames); 
%% Apply apodization and zero fills 
apodizationWindowFunction = @expwindow); 
F.data = apodize(F.data, F.parms, apodizationWindowFunction); 
zeroFills = 2; 
F.data = zerofill(F.data, F.parms, 2); 
%% Fourier transform and phase 
[S.data,S.ppm] = nmrft(F.data,F.parms); 
phaseObjective = @simplex_entropy; 
[S.data, S.phc0, S.phc1] = autophase(S.data, F.parms, phaseObjective); 
%% Remove unreal portion and align spectra 
X.data = realnmr(S.data, F.parms); 
X.ppm = S.ppm; 
X.data = icoshift(X.data, X.ppm); 
%% Save matrix as csv 
csvwrite("DataMatrix.csv", X.data); 
csvwrite("ChemicalShiftsData.csv", X.ppm); 
</code>

Example Scripts

2024-08-12T23:33:18Z

Mjeppesen: Created page with "=Example Scripts for Various MVAPACK Patterns= ''Note: Patterns may be sensitive to file location and path variables.'' ===Example 1: 1D NMR Processing to Matrix Export=== '''''%%''''' indicates comment line. All lines can be copied and pasted to terminal. <code> %% Start file extraction from 1D NMR files located in nmrdata sub directory filenames = glob("nmrdata/*"); [F.data, F.parms] = loadnmr(filenames); %% Apply apodization and zero fills apodizationWindowFunction..."

=Example Scripts for Various MVAPACK Patterns=
''Note: Patterns may be sensitive to file location and path variables.''

===Example 1: 1D NMR Processing to Matrix Export===
'''''%%''''' indicates comment line. All lines can be copied and pasted to terminal.

<code>
%% Start file extraction from 1D NMR files located in nmrdata sub directory
filenames = glob("nmrdata/*");
[F.data, F.parms] = loadnmr(filenames);
%% Apply apodization and zero fills
apodizationWindowFunction = @expwindow);
F.data = apodize(F.data, F.parms, apodizationWindowFunction);
zeroFills = 2;
F.data = zerofill(F.data, F.parms, 2);
%% Fourier transform and phase
[S.data,S.ppm] = nmrft(F.data,F.parms);
phaseObjective = @simplex_entropy;
[S.data, S.phc0, S.phc1] = autophase(S.data, F.parms, phaseObjective);
%% Remove unreal portion and align spectra
X.data = realnmr(S.data, F.parms);
X.ppm = S.ppm;
X.data = icoshift(X.data, X.ppm);
%% Save matrix as csv
csvwrite("DataMatrix.csv", X.data);
csvwrite("ChemicalShiftsData.csv", X.ppm);

Women in Science: Checklist

2024-04-03T19:04:57Z

Mjeppesen:

[[File:WomenInScience.jpg|thumb|Example setup of chemistry display table]]
==Two tables==
(provided by hotel)
#demonstration table
#recruiting material table

==Recruiting material==
#give away items
#candy
#fish bowls to hold candy (x2)
#poster display
#chemistry banner
#chemistry tablecloths
#powerpoint slide/video
#extension cords
#power strip
#projector

==Demonstration==
(request material from undergrad stock room two weeks before event)
#Oscillating clock
#Stop light reaction (can put more NaOH in)
#Orange Juice Clock
#Liquid Nitrogen Gummy Bear
#Lava lamps

== Miscellaneous Items ==
#A big empty used chemical container with cap
#A big beaker (1L)
#Nitrile gloves

[[category:Demos]]

File:WomenInScience.jpg

2024-04-03T19:04:01Z

Mjeppesen:

Women in Science: Checklist

2024-04-03T18:21:57Z

Mjeppesen:

[[File:files/misc/mediawikidownloads/WomenInScience.jpg|thumb|Example setup of chemistry display table]]
==Two tables==
(provided by hotel)
#demonstration table
#recruiting material table

==Recruiting material==
#give away items
#candy
#fish bowls to hold candy (x2)
#poster display
#chemistry banner
#chemistry tablecloths
#powerpoint slide/video
#extension cords
#power strip
#projector

==Demonstration==
(request material from undergrad stock room two weeks before event)
#Oscillating clock
#Stop light reaction (can put more NaOH in)
#Orange Juice Clock
#Liquid Nitrogen Gummy Bear
#Lava lamps

== Miscellaneous Items ==
#A big empty used chemical container with cap
#A big beaker (1L)
#Nitrile gloves

[[category:Demos]]

Women in Science: Checklist

2024-04-03T18:01:57Z

Mjeppesen:

Women in Science: Checklist

2024-04-03T18:00:29Z

Mjeppesen:

[[Image:WomenInScience.jpg]]
==Two tables==
(provided by hotel)
#demonstration table
#recruiting material table

==Recruiting material==
#give away items
#candy
#fish bowls to hold candy (x2)
#poster display
#chemistry banner
#chemistry tablecloths
#powerpoint slide/video
#extension cords
#power strip
#projector

==Demonstration==
(request material from undergrad stock room two weeks before event)
#Oscillating clock
#Stop light reaction (can put more NaOH in)
#Orange Juice Clock
#Liquid Nitrogen Gummy Bear
#Lava lamps

== Miscellaneous Items ==
#A big empty used chemical container with cap
#A big beaker (1L)
#Nitrile gloves

[[category:Demos]]

Women in Science: Checklist

2024-04-03T17:59:24Z

Mjeppesen:

[[File:WomenInScience.jpg]]
==Two tables==
(provided by hotel)
#demonstration table
#recruiting material table

==Recruiting material==
#give away items
#candy
#fish bowls to hold candy (x2)
#poster display
#chemistry banner
#chemistry tablecloths
#powerpoint slide/video
#extension cords
#power strip
#projector

==Demonstration==
(request material from undergrad stock room two weeks before event)
#Oscillating clock
#Stop light reaction (can put more NaOH in)
#Orange Juice Clock
#Liquid Nitrogen Gummy Bear
#Lava lamps

== Miscellaneous Items ==
#A big empty used chemical container with cap
#A big beaker (1L)
#Nitrile gloves

[[category:Demos]]

Women in Science: Checklist

2024-03-27T19:00:53Z

Mjeppesen:

==Two tables==
(provided by hotel)
#demonstration table
#recruiting material table

==Recruiting material==
#give away items
#candy
#fish bowls to hold candy (x2)
#poster display
#chemistry banner
#chemistry tablecloths
#powerpoint slide/video
#extension cords
#power strip
#projector

==Demonstration==
(request material from undergrad stock room two weeks before event)
#Oscillating clock
#Stop light reaction (can put more NaOH in)
#Orange Juice Clock
#Liquid Nitrogen Gummy Bear
#Lava lamps

== Miscellaneous Items ==
#A big empty used chemical container with cap
#A big beaker (1L)
#Nitrile gloves

[[category:Demos]]

Women in Science: Checklist

2024-03-27T18:59:26Z

Mjeppesen:

==Two tables==
(provided by hotel)
#demonstration table
#recruiting material table

==Recruiting material==
#give away items
#candy
#fish bowls to hold candy (x2)
#poster display
#powerpoint slide/video
#extension cords
#power strip
#projector

==Demonstration==
(request material from undergrad stock room two weeks before event)
#Oscillating clock
#Stop light reaction (can put more NaOH in)
#Orange Juice Clock
#Liquid Nitrogen Gummy Bear
#Lava lamps

== Miscellaneous Items ==
#A big empty used chemical container with cap
#A big beaker (1L)
#Nitrile gloves

[[category:Demos]]

Category:Maxey Demos

2024-03-27T18:48:43Z

Mjeppesen:

This is the category describing the demos for the Maxey Elementary School Chemistry Day.

Vitamin C in Juices

2024-03-27T18:48:23Z

Mjeppesen:

[[Category:Demos]]

=Experimental outline=

Determine the Vitamin C content in various freshly squeezed fruit juices. Compare kiwis and oranges (high concentration of Vitamin C) to peaches or apples (low concentration of Vitamin C). Or, blend a vegetable in water to create a solution that can be tested. Use cheesecloth or fiber to remove the fiber from the solution. Bell peppers are particularly high in Vitamin C but the green ones are supposed to have higher content than the red or orange ones. The Natural Hub has we page that lists the Vitamin C content of many fruits at: http://www.naturalhub.com/natural_food_guide_fruit_vitamin_c.htm

=Materials=

#Fruit juices and fruit drinks containing vitamin C (use 1.00 mL per experiment)
#Vitamin C Standard solution (1 mg/mL)(use 1.00 mL per experiment)
#Starch solution (sue 1.00 mL per experiment and control for 2.00 mL total)
#Iodine solution (we actually make I3-; need up to 1.00 mL per experiment, so need 2.00 L)

=Procedure for fruit juice or drink=

#Carefully squirt 1.0 mL of fruit juice or drink into a 10 mL graduate cylinder
#Carefully squirt 1.0 mL of starch solution in to the same 10 mL graduated cylinder
#Empty the contents of the cylinder into a small Erlenmeyer flask.
#Use a pipet bulb to add the iodine solution.
;Count the number of drops it takes to form a purple color.
Swirl the flask to make sure the solution stays purple. If it doesn’t, add one more drop.
#Report your number of drops to the assistant.
#Repeat the procedure with the vitamin C standard

=Procedure for vitamin C standard=

#Carefully squirt 1.0 mL of vitamin C standard into a 10 mL graduated cylinder
#Carefully squirt 1.0 mL of starch solution into the same 10 mL graduated cylinder
#Empty the contents of the cylinder into a small Erlenmeyer flask.
#Use a pipet bulb to add the iodine solution.
;Count the number of drops it takes to form a purple color.
Swirl the flask to make sure the solution stays purple. If it doesn’t, add one more drop.
#Report your number of drops to the assistant.
#You can calculate the mg vitamin C from the following equation:
;Amount of Vitamin C (mg)=(number of drops to fruit juice) / (number of drops to Vitamin C standard)

The equation above has been simplified from this equation
Estimated Vitamin C content = (1 mL) x (1mg/mL) x (number of drops to fruit juice) / (number of drops to Vitamin C standard)

Traffic Light Reaction

2024-03-27T18:48:06Z

Mjeppesen:

== Supplies ==

- Glucose:0.1M,250ml (take Glucose bottle)

- NaOH:3M,10ml (take NaOH bottle)

- Indicator: Indigo carmine

- 500ml Flask with a stopper (good stopper!) and glass rod for stiring

== Procedure ==

In a beaker containing 250 ml of distilled water, add 3 g of dextrose and stir until it dissolves. Next, add 5 g of sodium hydroxide pellets into the same beaker, and use a stirring rod to break apart the pellets so that they will dissolve quickly. Once this is done, pour the contents of the beaker into the glass flask, and add between 5-10 ml of the indigo carmine indicator. Enough should be added to the solution so that it turns a yellow color. Put the stopper in the flask, and let it sit for ten minutes. After ten minutes, the solution should be ready for the demonstration. Shake the flask once or twice, and it should turn a red color. Shake once or twice again, and it should turn green. Then, if it sits, it should turn back to the yellow color.

[[category:Demos]]

Spec20

2024-03-27T18:47:54Z

Mjeppesen:

==Purpose==

To find an unknown concentration of a solution by plotting known concentration of the same solution vs absorbance. A handout for the students is available here: [[File:Maxie_-_Spec20_Station_Handout.docx]].

==Experiment==

0. Preparation: Make sure the spectrometer wavelength is set to 590 nm. The instructions to calibrate the spectrometer is labeled on the front of each Spec20. Prepare six test tubes with percents ranging from 0% to 100% of blue dyes. Test tube 1 is water only it is to be used as the blank. Also every student should have one test tube labeled unknown.

1. Ask the students to draw two columns on a sheet of paper with PERCENT on top of column one and DATA on top of column two.

2. Ask the students to put test tube number 1(BLANK) into spec20. Check each station to make sure the absorbance reading is approximately is zero.

3. Ask the students to put test tube from number 2 to number 5 while reading off the reading corresponding percents 20%, 50%, 80% and 100% and tell them to record it in the PERCENT column as well as record the absorbance in the DATA column.

4. Ask the students to put the last test tube labeled "ev"(UNKNOWN) into the Spec20. Write down the absorbance reading in the data column and mark it as "x" in the percent column.

5. Inform the students to take the data sheets to the computer station.

[[Category:Demos]]

Peroxide Clock

2024-03-27T18:47:38Z

Mjeppesen:

1. Solution A: Prepare 100 mL of 9% H2O2 by diluting 30 mL of 30% H2O2 with 70 mL of deionized H2O.

2. Solution B: Prepare an acidified 0.2 M KIO3 solution by adding 10 mL of 1.0 M H2SO4 to 80 mL of deionized water. Dissolve 4.3 g KIO3 in this solution and dilute to 100 mL.

3. Solution C: Prepare starch solution by dissolving 0.1 g of soluble starch in 90 mL of boiling deionized water. When cool, add 1.5 g malonic acid, 0.4 g MnSO4·H2O, stir and dilute to 100 mL.

4. Add 50 mL of Solution A to a clean beaker fitted with a stir bar. Next add 50 mL of Solution B and let solutions mix thoroughly. Once complete, add 50 mL of Solution C and let reaction stir. Upon addition of the final solution, bubbles should appear. The solution will turn yellow then blue, then colorless. This reaction will oscillate for 5-10 minutes.

[[category:Demos]]

Orange Juice Clock

2024-03-27T18:47:21Z

Mjeppesen:

== Supplies ==

-Clock connected with two alligator clips

-Electrodes:Mg (negative end) and Cu (positive end)

-Ring stand + clamp

-Two beakers (250ml)

-Orange juice bottle

== Procedure ==

Put the two electrodes into a beaker with orange juice.

[[category:Demos]]

Maxey Day: Checklist

2024-03-27T18:46:18Z

Mjeppesen:

== Demos for Dr. Powers ==

'''1. OJ clock'''

- Clock set up, 500ML beaker, (x3) and electrodes (x2)

'''2. Elephants toothpaste'''

-Hydrogen peroxide(500mL 30%) , dish soap and KI (500 mL), Long cyclinder (x2), and a Nalgene tray to catch the foam

'''3. Red Cabbage and solid CO2'''

- Gallon of juice, a cooler of dry ice (night before), Large cyclinder with wooden base, and giant stir bar

'''4. Peroxide clock'''

- Hydrogen peroxide KIO3 and starch solutions with beakers (100mLx3), beakers (50mLx3), stir plate, and stir bar
'''
5. Glowing pickles'''

- Power supply and pickle
'''
6. Slides'''

- Projector, screen, extension cord, and power strips (Dr. Powers’ laptop)

'''7. Cryogens with banana and gummy bears'''

- Liq Nitrogen in 4L carrier, two foam buckets, a hammer, and cardboard set up to catch fly-away (Dr. J Powers brings the gummy bears and banana)

'''8. Stop light''' (We make these solutions)

- Glucose, Indigo Carmine, and NaOH. Get three 500mL Erlenmeyer flasks with good rubber stoppers from stock room.

----

== Outdoor Demos for Dr. Powers ==

'''1. Mentos and Coke''' (Dr. J Powers brings these supplies)

'''2. Potato Launcher'''

'''3. Isopropanol Rocket'''

-Demo room for 109/110 has this set up. Bring the Ignition

-Handwashing Station Demo for Dr. Joan Powers

'''1. Non-Newtonian fluids'''

-Corn Starch and water with drops of food color (make on site)

----

== Hands-on Stations ==

'''1. Nylon Experiment'''

-hexamethylenediamine and sebacic acid; (from Bob)

-10ml measuring cylinders (x50) atleast and 10mL beakers or paper cups (x100)

-Hooks to draw out the nylon polymer

'''2. Vitamin C in Juice'''

-Fruit Juices (Dr. J Powers brings these)

-Starch solution (1 gallon) (make the night before)

-Iodine solution ++ (1-2 L)

-10mL beakers (x50) and 50mL beakers (x20) plastic droppers (x1 box)

'''3. Batteries'''

-Pennies

-Meters (as many as working)
'''
4. Blue Dye UV-Vis
'''
-Spec 20s (from 109 Labs ask Lab manager for these)

-Plastic cuvettes

-Blue dye and their dilutions (1L) each

-Beakers (10mL x 20)

'''5. Computer Stations'''

- Printer

- Paper

- Laptops (x5)

- Cords and extension cables

'''6. Slime making'''

- Glue (2 gallons 50%)

- Borax saturated (1 gallon)

- Paper cups

- Wipes

- Zip lock bags to take along

- Food colors

[[category:Demos]]

Making Nylon

2024-03-27T18:45:53Z

Mjeppesen:

The process of making nylon involves the combination of hexamethylene diamine with sebacoyl chloride. This particular procedure is producing what is known as Nylon 6-10.

== Materials ==

:* 1,6-hexanediamine in water (> 750 mL)
:* 20% NaOH (100 mL)
:* 5% sebacoyl chloride in hexanes (> 750 mL)
:* 100-mL beaker (90 - 100 beakers)
:* 50-mL beaker (10 beakers)
:* Plastic dropper (10 droppers)
:* Thin wire (1 small spool)
:* Two 10-mL graduated cylinders (20 graduated cylinders)
:* Paper towels
:* Goggles and gloves

== Intro for the Students ==

:* Ask them if they know what nylon is.
:* Tell them where they’ll find nylon.
:* Nylon is one of the first synthetic substances. Ask them what "synthetic" means.
:* They are going to make nylon from two chemicals.
:* Nylon is a polymer which means each chemical stacks on each other like building blocks to make the strand of nylon.
:* They will see the two chemicals merging together to form a strand of nylon in real time.

== Procedure ==
=== Setup ===

# Give each group a clean 100-mL beaker. This beaker will be replaced for each new group.
# Each group should have a small 50-mL beaker with about 20 mL of 20% NaOH already in it. Place a plastic dropper near or in the beaker. This beaker should last the entire day since each group only uses 2 drops worth.
# Each group should have enough small pieces of wire (3-4 in.) for each member of the group. The wire should have a small bend at the bottom to help it hook onto the nylon. Usually have to cut the wire from a small spool.
# Each group should also have two 10-mL graduated cylinders. You will need a way to distinguish between the graduated cylinders. Different colored tape or plastic bases would be the best way to do this. Don’t use markers since the organics will just wipe it off.
# At the beginning, you will need to pour out the chemicals into the graduated cylinders for them. Do NOT let them measure it out themselves. Into one graduated cylinder, pour 3-4 mL of 1,6-hexanediamine, and to the other pour 3-4 mL of sebacoyl chloride.

=== Procedure for Students ===

# Pour the contents of the graduated cylinder containing the 1,6-hexanediamine into the 100-mL beaker in front of them.
# Take 2 drops of the 20% NaOH from the small beaker and add it to the 100-mL beaker.
# SLOWLY pour the contents of the graduated cylinder containing the sebacoyl chloride into the 100-mL beaker in front of them.
# Before letting the students pull out the nylon, have the students look closely at the beaker for any visual changes. They should see a faint white skin forming on top of the liquid.
# Have the students use the wires to hook the nylon skin and start SLOWLY pulling it up. Have them try to see how high they can make it go. Let them stand on their chairs but not the table. Have the other members of their group help keep the nylon string going. Be sure to ask the student what’s happening at the surface of the liquid.
# If the nylon breaks, it will restart if you put some of it back in the liquid and start pulling up again.
# Feel free to let the students touch the nylon with their hands as long as they have gloves on. Ask them what it feels like.
# When the liquid is gone, the nylon strand is done. Have the students place the nylon back in the beaker and have them wash their hands/gloves off.

=== Finishing ===

# Place the used 100-mL beakers back in the box and grab new ones.
# Wipe off any excess nylon residue from the wire.
# Refill the graduated cylinders with the appropriate chemicals as above.

== Notes ==

:* Lead the entire group through the steps. Have each group do the first step and wait until they all have done it before moving on to the next step and so on. Life is much easier that way.
:* Don’t worry about telling them the names of the chemicals. Just tell them which color graduated cylinder to pour in.
:* The students will always ask if they can step on the table, don’t let them.
:* Remind the students not to get the nylon on their skin or clothes.
:* Make sure to keep the organics capped at all times especially since they evaporate so quickly.
:* Keep a lot of paper towels nearby.
:* In the past, we always come close to running out of either 100-mL beakers or organics. Make sure that you have at least 90-100 beakers and no less than 750 mL of each organic.

[[Category:Demos]]

Gummy Bear Freeze

2024-03-27T18:45:35Z

Mjeppesen:

== Supplies ==

- Liquid nitrogen

- Gummy bears

- Hammer

- A folded box as supporter of smashing

- Tong to hold gummy bear

- Safety glasses, thermal gloves

- Bucket (hold liquid N2)

- Tape

== Procedure==

Bring all supplies to demo and set up for gummy bear smashing. Make sure to clean up bummy bears immediately after demo is finished - much harder to clean after gummy bears have thawed.

[[category:Demos]]

Computer station

2024-03-27T18:45:04Z

Mjeppesen:

At the computer station, students will graph the data points they collected from the Spec20 station and draw three molecular structures. The handout to be given to students is available here: [[File:Maxey_-_Computer_Station_Handout.docx]].

== Preparation ==

''This should be ready the day before)''

* Six to eight laptops should be provided and they are all set in a classroom in the Maxey elementary school.
*Laptops can be checked out from the 501 building; they are general use student laptops. E-mail Wade Felker (Collaboration Technologies Associate with ITS; wfelker2@unl.edu), a week in advance to reserve as many laptops as you need.
* If the laptops don't give you admin access to install software: (Set up an account for Maxey demo for each laptop.)
* Install ChemBioDraw and KaleidaGraph software on each laptop. (We have licenses for both pieces of software; ask Eli.)
* Set up wireless printer/router for each laptop. (You can also set them up as a wired system if all else fails)

== Experiment A - KaleidaGraph ==

KaleidaGraph is used for processing the Spec20 Data. ''(Make sure the students finish the Spec20 experiment before they come to this station.)''

# Click the first white box in the toolbar and you can open an empty spreadsheet with three columns.
# Input the data of concentration and absorbance in column A and B. (Do not input the percentage symbol or the unknown sample absorbance.)
# Go to “Gallery" from the menu, click “linear” then “scattering”, then there is a window popping up. Choose the A column to be the X-axis, B column to be the y-axis and click “new plot” icon down below.
# After you have the plot, change the title to be "Absorbance vs. concentration"
# Go to “file” and click “print graphics”.
# After the students print out their graph, you may want to help them draw a trendline manually using a ruler. Then you can demonstrate determining the concentration of their unknown using this trendline.

== Experiment B - ChemDraw ==

*NOTE: You usually don't have time to get to this station. Use your best judgement, and go on if the students have extra time.

Three molecules will be drawn by using ChemBioDraw: Acetic acid is the main component of vinegar. Acetylsalicylic acid is the active reagent of aspirin. Glucose is the major energy source in our body.

On the left side of the working window, a toolbar contains all the tools that are handy for drawing those molecules, such as single bonds, double bonds, benzene, texting and eraser.

# Find an empty place and draw the first bond by dragging the icon of a bond from the toolbar to that place and loose your mouse.
# Move the mouse to the end of that bond where you want to start another bond or a word (for example, "H" is for proton or hydrogen. "C" is for carbon and "O" is for oxygen).
# After drawing the molecule, use the “lasso” from the toolbar to select the whole molecule, and then go to the menu and select “convert structure to name”. Find out if you do the right thing by comparing the name that the computer tells you with the name you are supposed to draw.

[[Category:Demos]]

Coin battery

2024-03-27T18:44:26Z

Mjeppesen:

[[Category:Demos]]

==Materials:==

A small box of coins (quarters, dimes and nickels)

Saturated potassium chloride (200mL)

Filter paper strips (>100)

Voltage meters (5-10)

Paper towels

Goggles and gloves

==Intro for the students:==

Ask the students about batteries in daily life

Explain the function and usage of voltage meter

Explain why the stack of coins can produce electronic current

==Procedures (for each group):==

a. Soak the filter paper strips in saturated potassium chloride.

b. Stack two pile of coins with various combination.

c. Put the two piles together with the filter paper in between.

d. Measure the voltage of the whole stack using a voltage meter.

e. Repeat to get the highest voltage value by changing the order and combination of coins.

==Notes:==

a. Make sure all the voltage meters are working before use. If not, change the battery.

b. Make sure the alligator clip from the voltage meter has a clean metal surface. If not, use sand paper to remove the rust.

Checklist electronic shop

2024-03-27T18:43:54Z

Mjeppesen:

Laptop X 1

Laptop charger X 1

Laptop lock X 1

LCD projector X 1

Router X 1

Ethernet cable X 1

Voltage meter X 1

Speaker X 1 pair

Electric extension cable and board X 8

Troubleshooting

2022-07-21T20:40:43Z

Mjeppesen:

There a number of problems that you might encounter on your workstation. This page is an attempt to catalog the most commonly encountered problems, and fixes as they are found.

This list is by no means exhaustive, and may not contain a solution for every problem.

Windows
-Mouse and Keyboard Not Working?
--This can happen pretty frequently, especially if you start building new workstations. Most of us use wireless USB keyboards, and newer motherboards have options in their BIOS to enable/disable drivers for USB 3.0 ports. If these options exist, you'll generally want XHCI Hand-off enabled, and EHCI hand-off DISABLED. This is particularly relevant if the keyboard and mouse work in the BIOS and GRUB but not in Windows.
Link: http://superuser.com/questions/770198/should-i-enabled-or-disable-xhci-hand-off-in-bios-setup-when-running-windows-7

Linux

Updates not working Correctly? Check your sources list. Some of us are running Debian 7 (wheezy) and some are on Debian 8 (Jessie). If you notice that updates are not working, or dependencies can't be resolved because of broken packages, it could very likely be your sources list. An excellent discussion can be found on the debian website.
Link: https://wiki.debian.org/SourcesList

[[category:Troubleshooting]]

99p

2022-07-21T20:40:26Z

Mjeppesen:

There a lot of problems that arise whenever you start relying on computers for your work.

Here are 99 random problems you'll probably never run into. When you do though, you'll be glad you have this list.
Solutions to various problems will be kept here until a more permanent resting place is found.

nmrDraw Troubleshooting:
Having problems with a "segmentation fault" error? Especially if you're on one of the newer workstations?
Try checking your memory limits.
Type "ulimit -a" for information. The number of open files should be set to 1024. You can do this by typing "ulimit -n 1024".
If it is higher, you'll almost always get a segmentation fault. This was first discovered on macs, but it also holds true for linux based systems.
EDIT: "ulimit -n 1024" only works for the currently open terminal window. It will reset as soon as you open a new terminal.
If you want to set this limit permanently, you can add the function to your .bashrc file in your home directory. This will set the limit in every new terminal window you open.

Source: https://beta.groups.yahoo.com/neo/groups/nmrpipe/conversations/messages/2603
[[category:Troubleshooting]]

Category:Troubleshooting

2022-07-21T20:40:09Z

Mjeppesen: Created page with "category:Protocols"

[[category:Protocols]]

'''Check in for the NMR 700mHz'''

2022-07-21T20:39:27Z

Mjeppesen:

Make sure the system is accessible, e.g. not being used or under maintenance

Check your belongings on your body and remove anything with ferromagnetic properties such as electronic devices and keep them away from the probe

Go to the Sample Jet and change the operating mode to 5mm shuttle, then load the rack of NMR tubes.

Login to the computer

Start topspin software

Load sample by typing in “sx 101” (or if in rack 2: “sx 201”, etc)

Click on the blank document icon

*1. Name it: initials-title-date
*2. Experiment: zgesgp (wipes out the water peak)
*3. Set solvent
*4. Check the “getprosol” box

Lock the sample by typing “lock d2o”
*1. The signal should be at around 75% after you lock it. If it is too low, go to BSMS, click “power” and use the scroll on the mouse to get it to around 75%, then click “standby” on BSMS to save the change.

Shim the sample automatically by typing “topshim”
*1. If the signal is not at 75%, adjust it in the same way as before. Also you can adjust the shimming manually by going to the BSMS and clicking on “z1”, “z2”, “x”, “y”, “xy”, etc. dimensions and using the scroll on the mouse. Don’t forget to click “standby” after any changes.

Autotune the sample by typing “atma”. To manually autotune, type “atmm”.

Next you need to find the 90° pulse.
*1. Type P1 and enter “8.0” in the pop-up window
*2. Type “pulprog” and choose “zg”
*3. Type “ns 1” (number of scans=1), then type “ds 1” (dummy scans), then type “rg 1” (receiver gain).
*4. Run the sample by typing “zg”
*5. To transform FID into spectrum, type “efp”
*6. Type “apk” to autophase
*7. Not type “p1”, hit enter and try different numbers (usually between 30-44), type “zg” and “efp” in order and see what number (p1) minimizes the peak the most.
*8. Once you have found the p1 number that minimizes the peak, divide that number by 4. That number is your 90° pulse.
*9. Type “edprosol” and set the p1 you found into both pulse widths and hit enter in each one
*10. Click the “copy to solvent” buton and select all relevant
*11. Click the “copy to probe” and select all relevant again. A popup window will ask you which cryoprobe you want to use. Choose the one that matches the cryoprobe that is in the top left corner.
*12. Save then “select all relevant”. Click on “yes” and “ok” whenever it asks you and make sure that you calculate all pulses when it asks. Then close out.

To run IconNMR, type “iconnmr” and choose the automation option and enter the password

Go to the holder that your sample is in. For example if your sample is in the 1st rack and is in the first slot in that rack, then go to A1-101.

Type in the name of your experiment, click on the “No.” space to activate that box, choose your solvent, and choose your experiment.
*1. Experiment for HSQC: C13HSQCS1SP2 (metab)
*2. Experiment for HMBC: C-13 HMBC
*3. Experiment for HSQC-TOCSY: SL-hsqcetf3gpml. Metab

Click on the paramters tab and choose “edit all acquisitions parameters”
*1. In the TD (data points) for the H dimension enter a number that is between 1024 and 2048 although it’s usually 2048. In the C dimension enter a number that is between 64 and 256 although it’s usually 64.
*2. In the NS (number of scans), type 32 although it can be 64 and even 128 depending on what you want.
*3. Type d1 and hit enter and input 1.5 for the relaxation time. *If you are doing the HSQC-TOCSY experiment then make sure you also change the d8 (delay time) to whatever number corresponds to the right hand panel
*4. Type in “rga” to check the automatic receiver gain
*5. Go to automation tab and make sure that the AUNM is set to au-zgonly.
*6. Then click on the button that says “return to iconNMR”

Copy to the samples as needed. (“edit as needed” )

Highlight all of the samples and click submit

Click “Start” and check the “lock/shim has already been completed box” and make sure that you are starting at your sample (for example: 101).
[[category:NMR_Usage]]

Working with deuterated solvents

2022-07-21T20:39:01Z

Mjeppesen:

# Turn on gas cylinder using main regulator
# Adjust with secondary regulator so that max PSI is about 150
# Place deuterated solvent under the nozzle and open cap
# Open and adjust tertiary regulator so that air flow is strong enough to displace headspace in the bottle but not so fast that things splash out
# Keep bottle under nozzle when not using it
# When finished tighten and seal bottle and turn off cylinder at main regulator
# Close tertiary regulator
# Close Hood

=== Important Notes ===
* Deuterated solvents are expensive and must be treated with care to prevent exchange with H2O in the air.
* Remember to tighten and seal all deuterated solvent bottles after each use

[[category:Sample_Preparation]]
[[category:NMR_Usage]]

Water-Suppression using pre-saturation pulses (zgpr/zgcppr)

2022-07-21T20:38:41Z

Mjeppesen:

==Justification==
The majority of samples we run in D2O suffer from residual H2O signal masking the actual small molecule signals. This is because the small molecule concentration is typically ~100μM unlike water which has a concentration of 55M so any residual protons which have exchanged with the D2O will overpower any small molecule signal that is not of sufficient concentration. However, we can get around this issue using water suppression methods. A low power pulse at the solvent frequency and is applied during the preparation delay. This low power pulse excites the water proton signal such that no signal can fully accumulate and be measured.
===Water-Suppression===
[[Image:Water_suppression_spectrum.png]]

The figures above displays a no water suppression zg30 spectrum compared to a water suppression zgpr spectrum respectively.

===ZGPR/ZGCPPR Pulse Sequence===
[[File:ZGPR_ZGCPPR_pulse_sequence.png]]


Both methods are comparable in data quality, experiment time and will suppress residual water signals. The benefit of the zgcppr pulse sequence is a more complete saturation by compensating for inhomogeneities in the applied B1 field using a series of composite 90o pulses. The result is a slightly more narrow residual water signal that must be removed during processing (zgpr: 265Hz zgcppr: 221Hz) and would be beneficial for molecules with resonances near that of water.

[[Image:ZGPR_ZGCPPR_spectrum.png]]
==Methodology==

====Finding the O1====
NOTE: The commands are for Bruker software primarily TOPSPIN.

NOTE: After any command is typed you must press enter to execute.

# We must first check where the residual water peak resonance appears in the spectrum so that we know where to apply the low power pulse to suppress the signal, this is called the O1. More generally the O1 is the center of the spectrum and is used as a reference.
# Follow steps to setup (create file, lock, shim, tune etc…) a typical 1D experiment
# Once the experiment is set up type 1H in the command line and press enter (NOTE: This will pull in the standard parameters for a typical 1D spectrum (no suppression) using the zg30 pulse sequence)
# Set number of scans (ns) to 1: Type ns and set to 1
# Set number of dummy scans (ds) to 0: Type ds and set to 0
# Set the receiver gain: Type rga (NOTE: rg should be about 10-35 check by typing rg)
# Run spectrum: Type zg
# To view FID type a
# When finished acquiring data Type efp to process
# Auto phase correct: Type apk
# Auto baseline correct: Type abs
# Move cursor over center of water peak (Very large peak ~4.5-5.0ppm) write down peak center in Hz
# Type iexpno to advance to the next experiment file
====Water Suppression Pulse ====
# Under the new experiment file: Type pulprog and enter in the experiment you want to run (zgpr/zgcppr)
# Enter the solvent that you are using (typically D2O) Type solvent
# Pull in the standard pulse parameters (i.e. pulse lengths and delays) Type gpro (NOTE: You must have the correct solvent set to enter correct parameters)
# Type O1 and enter in new O1 value in Hertz (Hz) from the above experiment
# Set receiver gain Type rga (NOTE: rg should be about 35-50 check by typing rg)
# Set initial number of scans to 1 Type ns and set to 1
# Set initial number of dummy scans to 0 Type ds and set to 0
# Set delay/pre-saturation length: Type d1 and set to 2.00seconds (WARNING: The Cryoprobe is expensive!!! We don’t want to burn out the probe by putting in too much power. When using the Cryoprobe DO NOT set d1 above 2.00seconds with a power level no greater than 50dB!!!)
# Type zg (Note: this short experiment will let you take a quick check on the quality of water suppression)
# If water suppression is acceptable (i.e. minimal water signal) set ns and ds back to desired parameters (NOTE: Typically for our runs at 100μM small molecule ns=128 and ds=16)
# Type zg

When finished running SIGN LOG BOOK.

Transfer data to workstation or onto computers in the lab with ACD software.

[[category:Data_Collection]]
[[category:NMR_Usage]]

Urine Preparation for 1D NMR

2022-07-21T20:38:25Z

Mjeppesen:

1. Urine samples are aliquoted and centrifuged at 14,000 × rpm for 10 min to pellet debris

2. Supernatant samples are collected and snap frozen in Liquid Nitrogen and freeze dried to remove methanol by speed vacuum centrifugation (SpeedVac R Plus, Savant) and water by lyophilization using FreeZone™ (Labconco, Kansas City, MO) for 24 hours and stored at -20°C until NMR and MS analysis.

Note: Methanol extration used in serum preperation can be used to minimize protein content and urea peak in NMR analysis.

[[category:Sample_Preparation]]

Tube deep cleaning

2022-07-21T20:37:40Z

Mjeppesen:

[[Category:General_Maintenance]]
[[Category:Metabolomics]]

1. Empty all contents from dirty NMR tubes into appropriate residue container. Remove the labels on the NMR tube by rinsing with ethanol.

2. Fill the container outside the protect bottles with liquid nitrogen and cool it down.

3. Load the tubes into the tube cleaning apparatus (tube washer) with proper solvents as noted below.

4. The operation of the tube washer:

a)With the left valve closed, slowly and briefly opening the T-valve to the vacuum position evacuates the air from the Labconco®
vacuum bottle and places it under a slight vacuum without removing the solvent.
b)Turn the T-valve to the vent position opens the vacuum bottle to atmosphere breaking the vacuum and forcing solvent into the NMR tube.
c)Switch the T-valve back to the vacuum position places the NMR tubes back under vacuum and remove the solvent from the NMR tube.
d)Repeat the process two or three times effectively washes the NMR tubes with solvent.
e)Switch the T-valve to the vent position and opening the left valve rapidly removes the solvent from the NMR tube cleaner into the
filter flask. The process can be repeated with other solvents.

5. Rinse NMR tubes with nano pure water by filling with water then empty all contents from dirty NMR tubes into appropriate residue container.

6. Rinse NMR tubes with ethanol by filling with ethanol then empty all contents from dirty NMR tubes into appropriate residue container.

7. Rinse NMR tubes with acetone by filling with acetone then empty all contents from dirty NMR tubes into appropriate residue container. The amount of organic solvents should be well-controlled.

8. Fill tubes with nano pure water.

9. Place NMR tubes in a concentrated nitric acid bath and soak 24hrs.

10. Empty contents of NMR tubes into the acid bath.

11. Rinse the NMR tubes under the tap water to remove most of the acid.

12. Load the tubes back to the tube washer.

13. Rinse NMR tubes in baking soda solution to neutralize acid.

14. Rinse NMR tube thoroughly with nano pure water until the solvent waste is neutral.

15. Dry NMR tubes: lay tubes horizontally in the oven.

16. Once dry inspect tubes for any crack, divots or other defect. The tubes with crack on the opening should be collected for further modification and reuse. Other problematic tubes have to be disposed.

[[File:Tubewasher1.jpg]][[File:Tubewasher2.jpg]]

A demonstration video can be found at here [http://bionmr.unl.edu/files/misc/tubewasher-instructions.wmv]

Serum Preparation for 1D NMR

2022-07-21T20:36:28Z

Mjeppesen:

1. Serum sample are first aliquoted

2. Proteins are precipitated by the addition of twice the amount of methanol as sample

3. Antibacterial NaN3 is added 2.0% by volume to avoid any bacterial growth

4. The 1:2 mixture of serum: solvent is then vortexed for 10 seconds

5. Samples are incubated at 4°C for 10 minutes

6. Next, the samples are centrifuged at 14,000 rpm for 20 mins at 4°C to pellet the proteins

7. The supernatant is collected and centrifuged at 14,000 rpm for 5 min a 4°C to pellet any leftover protein

8. At this point, the supernatant samples are collected and snap frozen in Liquid Nitrogen and freeze dried to remove methanol by speed vacuum centrifugation (SpeedVac R Plus, Savant) and water by lyophilization using FreeZone™ (Labconco, Kansas City, MO) for at least 24 hours and stored at -20°C until NMR analysis.

[[category:Sample_Preparation]]

Protein Overexpression and Extraction

2022-07-21T20:36:14Z

Mjeppesen:

Protein Overexpression and Extraction
- See also [[Protein_expression | Protein Expression]]

# Prepare and sterilize a minimal media where the carbon and nitrogen source can be controlled (M9 media). Make sure the volume of the media does not exceed half of the total volume of the flask that holds it (ex. 1L of media in a 2L flask).
# Add the appropriate drug to your media to select for your bacteria and plasmid.
## 100mg/L ampicillin
## 30mg/L kanamycin
# Inoculate the media with your bacteria.
# Allow the culture to grow to an A600 of 0.7-0.8.
# Induce protein overexpression by adding IPTG to a final concentration of 0.4-2mM. Note the time.
## Start with 0.4mM (~0.1g/L). High concentrations of IPTG may be toxic.
# Protein production should be monitored over 5 hours. At 1 hour time intervals, take a small amount of culture and save for SDS-PAGE analysis.
## Future cultures can be harvested at the time when most of the target protein is present.
# To harvest, spin the cultures down in the refrigerated centrifuge.
## I’ve found that 20 min at 4,000 rpm is sufficient.
# Resuspend the bacteria in no more than 25 mL of lysis buffer.
##Optional (but recommended): Add a protease inhibitor cocktail or a spatula tip of phenylmethanesulfonyl fluoride.
##Optional (but recommended): Freeze/flash freeze the resuspended cells and keep them in the freezer overnight. This helps to lyse the cells.
# Allow the cells to thaw and bring to room temperature.
##Optional: Add 1mg of lysozyme per gram of cell pellet. Rock at RT for 20-30 minutes.
# Sonicate until the cell suspension changes color. It usually clears up or becomes a milky color.
## 10 bursts (10 sec) on a high power setting.
# Spin down the cell debris.
## 20 min at 4,000 rpm.
# The supernatant contains protein. Save the pellet in case your protein is stuck in an inclusion body.

[[category:Protein_Preparation]]
[[category:Cell_Culturing]]

Preparing Protein for FAST-NMR

2022-07-21T20:36:01Z

Mjeppesen:

This page will describe the process by which proteins that are to be screened by FAST-NMR are prepared, tested, and added to the database.
[[category:FAST-NMR]]

Plasmid Purification and Transformation Protocol

2022-07-21T20:35:41Z

Mjeppesen:

Plasmid Purification/Transformation Protocol – JC 02/18/2016
Adapted from protocols found at “http://web.mnstate.edu/provost/”

# Expand culture of bacteria overnight in 5-7mL of media with antibiotic (has plasmid).
# Spin down culture for 10min. Turn on water bath and set to 42˚C.
# Remove supernatant.
# Resuspend pellet in 750uL of Buffer P1 with RnaseA (Ok to vortex).
# Add 750uL of Buffer P2, mix. Incubate @RT for 5min (DO NOT VORTEX).
# Add 750uL of chilled Buffer P3, mix. Incubate on ice for 15min (DO NOT VORTEX).
# Centrifuge for 30min (Do this in the refrigerated centrifuge).
# Save supernatant in a clean tube.
# Repeat Step 7 if particulate matter remains.
# Transfer 1mL to two microcentrifuge tubes and add 700uL of isopropyl alcohol to each, mix.
# Centrifuge at max speed for 30min.
# Rinse pellet with 70% ethanol and centrifuge for 30min. The pellet may not be visible.
# Let pellet dry for 10-20min and resuspend in 75uL Tris-EDTA buffer (TE).
# To 50uL of competent cells, add 5uL of plasmid (from 12).
# Incubate on ice for 30min.
# Heat shock at @42˚C for exactly 30sec.
# Add 250uL of Super Optimal Broth + 20mM Glucose (SOC).
# Spread all of it on a prewarmed LB plate with antibiotic.
# Allow plate to dry (in the laminar flow hood) and incubate inverted @37˚C overnight.

See also [[Plasmid_Transformation | Plasmid Transformation]]

Recipes
P1
* 50mM Tris base 6.06g
* 10mM Na2EDTA.2H2O 3.72g
* HCl to pH 8
* H2O to 1000mL
* RNAseA 50ug/ml just before use
P2
* 200mM NaOH 8g
* 1% SDS 10g
* H2O to 1000mL
P3
* H2O 500mL
* Potassium Acetate 294.5g
* Glacial Acetic Acid 110mL
* H2O to 1000mL
TE
* 10mM Tris
* 1mM EDTA
* HCl to pH 8
SOC media
* 2% tryptone 20g
* 0.5% yeast extract 5g
* 8.56mM NaCl 0.5g
* 2.5mM KCl 0.186g
* H2O to 1000mL
* 10mM MgCl2 0.952g
* 10mM MgSO4 2.467g
* 20mM glucose 3.603g

[[category:Protein_Preparation]]
[[category:Cell_Culturing]]

MetPa for metabolomics

2022-07-21T20:35:26Z

Mjeppesen:

*To use metpa for metabolomics pathway analysis go to http://metpa.metabolomics.ca/MetPA/faces/Home.jsp
*enter the list of metabolites using Keeg or HMDB id (using metabolite names is sometime confusing)
*select the pathway library according to the organism of interest
*select the overepresentation and topology analysis
*submit the data
[[Category:Metabolomics]]

Metabolite Extraction

2022-07-21T20:35:11Z

Mjeppesen:

==Protocol 1.1 (''Staphylococcus_Filtration'')==

Intro:
An optical density (O.D.) 600 of 10 was achieved for the total number of cells collected for one dimensional (1D) 1H NMR, or an O.D. 600 of 20 was collected for two dimensional (2D) 1H-13C HSQC NMR experiment. To reach targeted O.D. 600, 14-16 mL of culture was used for each sample for three hour growth and 3-5 mL was used for eight hour growth.

Steps:

1. Millipore® Microfil ® V filtration system (0.45 micrometer pore size, pre-sterilized filter paper) was used to separate cells from the growth media. Each filter paper was pre-washed immediately before use, with 10 mL 20 mM phosphate buffer (pH 7.2) and was used for collecting one culture sample.

2. After the filtration of bacteria cells, the filter membranes were placed into 50 mL Falcon tubes (pre-cooled at -70oC).

3. To quench the cells, the tubes were sealed, and put into a bucket of liquid nitrogen immediately.

4. The cells were transferred to a 1.5 mL tube (pre-cooled at -20°C) from each filter membraneindividually by suspension with 1 mL ice-cold 20 mM phosphate buffer.

5. The cells were spun down at 13200 rpm for one minute at -9°C to remove the residue media, and were re-suspended in 1 mL ice-cold phosphate buffer.

6. The O.D. 600 (1:100 - 1:200 dilution) of the cells from each sample was measured and adjusted to a targeted O.D. 600 in 1 mL phosphate buffer.

7. FastPrep® system (MP Biomedicals) was used to lyse the cells. The cells from each sample were disrupted by glass bead in a 2 mL tube (pre-cooled at -20oC) twice following a pre-set cycle (Speed 6, 40 s, Program 1) with a 5 minute rest time.

8. The tubes were centrifuged at 13200 rpm for two minutes at -9°C.

9. 700 µL supernatantin each tube was transferred into another 2 mL fresh tube (pre-cooled at -20°C).

10. An extra washing step for the cell debris with 1 mL ice-cold phosphate buffer was performed and 900 µL of supernatant was taken and pooled with the previous 700 µL extraction.

11. A final 1.5 mL sample in each fresh tube was prepared without any glass beads.

12. All tubes were flash frozen in liquid nitrogen and then stored in dry ice for a short transportation period.

13. The samples were lyophilized completely. 600 µL D2O was added to the lyophilized samples. 50 µM TMSP for 1D 1H NMR or 500 µM TMSP for 2D 1H-13C HSQC NMR was also added to each sample as an internal standard. The samples were held in 5 mm NMR tubes (Norell ST500-7, Norell, Inc., Landisville, NJ USA).

==Protocol 1.2 (''Staphylococcus_Centrifugation'')==

1. Streak the plates for individual colonies.

2. Pick up one colony for each test tube of 3 mL TSB media and inoculate it for a day (~9 hours). Do necessary repeats with different cultures.

3. Titrate 15 µL pre-culture being grown during the day into 3mL TSB for overnight 12 hour growth for each culture.(1:200 ratio dilution).

4. Use UV/Vis spectrometer to find out O.D. Dilute the sample into 1:20 when necessary to keep the O.D. value in its effective range of reading.

5. For 2h growth, in order to have enough cells, 36 mL TSB media is used and for 6h growth, 18 mL TSB media is used. We have two types of flasks: 125 mL and 250 mL. So 18 mL media is kept in the smaller flask and 36 mL media will be held in the large flask. In both cases, the ratio of the media and the total would be 1:7 so that we will have enough oxygen during the growth.

O.D. and pH after growth are recorded.

6. All growths are in constant 37oC.

7. Lysate:40ms, program 1 Spin down: 5 min maximum speed (132000 rpm)

8. Centrifuge: rotor->29->SH-3000BK->4000->12 min

==Protocol 2 (''Mycobacteria_Centrifugation'')==

1. All bacterial culture were grown in 50mL of Middlebrook 7H9 media for roughly 14 hours at 37oC with shaking at 200 rpm until an OD600=0.6 was achieved.

2. The cultures were placed on ice for 5 minutes, and centrifuged for 10 minutes at 2,700 rpm.

3. The used media was removed and the cell pellets were washed three times with 30 ml of ice cold double distilled water.

4.The washed cell pellets were resuspended with 10 ml of double distilled water and transferred to 30mL Pyrex beakers.

5. The cell pellets were then sonicated on a salt-ice water bath with a Vibra Cell Model VC600 for 5 minutes in the presence of 30% (vol/vol) type A-5 alumina.

6. The cells were centrifuged for 30 minutes at 15,000 rpm, and the supernatant was collected to remove any cell debris.

7. The supernatant was transferred to a 50ml Corning tubes and frozen in an ethanol-dry ice bath and stored at -80oC until ready to be analyzed.

8. The supernatant was lyophilized and resuspended with 700 uL of 99.8% D2O containing 50 mM phosphate buffer (pH=7.2) with 50 uM of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP).

9. The samples were vortexed and centrifuged for 3 minutes at 13,000 rpm, and 600 uL of the cell free extract was transferred to NMR tubes.

==Protocol 3 (Pancreatic cancer and Neuroblastoma cells_''MeOH extraction'' )==

1. Aspirate off medium.

2. Tilt plate, aspirate of all media.

3. Keeping plated tilted, wait a few seconds to allow any additional media to collect in corner of plate, and them aspirate to get off as much as possible.

4. Add 5 mL of phosphate buffer and wash twice.

5. Immediately add 1 mL of 80% methanol (-80°C).

6. Place the plates at -80 °C for 15 minutes.

7. Scrap plates on dry ice with cell scraper.

8. Transfer cell lysate/methanol mixture to 2.5 mL eppendorf tube.

9. Centrifuge at full speed for 5 minute in cold room to pellet cell debris and proteins.

10. Transfer the supernatant to 2.50 mL eppendorf tube.

11. Re-extract the cell pellet with 0.25 mL of water.

12. Add to the sup collected from 80% methanol extract.

==Protocol 4 (Parkinson disease cells_''MeOH extraction'' )==

==Protocol 5.1 (Biofluids_urine)==

[[category:Sample_Preparation]]
[[category:Metabolomics]]

Media preparation

2022-07-21T20:34:38Z

Mjeppesen:

[[Category:Protein expression]]
[[category:Cell_Culturing]]
===1. General Protocols For Minimal Media Protein Expression===

Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source for 1L 5xM9 salts:

64 g Na2HPO4-7H2O

15 g KH2PO4

2.5 g NaCl

H2O to final 1L volume and autoclave
To prepare 500 mL M9 minimal media:

100mL of 5xM9 salts:

1 mL 1 M MgSO4

50 uL 1 M CaCl2

5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)

2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5 g dry 10 mL 20% d-glucose or 2 g dry

Glass distilled & autoclaved H2O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).

Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/mL (or any antibiotics used to select the strains).

Grow 5mL overnight culture in same media to inoculate 500mL M9.
Shake culture at 37 oC until an OD600 of 0.7
± 0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).

===2. General Protocols For LB Media===

For 1 L media preparation

10 g Bacto-tryptone

5 g yeast extract

10 g NaCl

Sterilized by autoclave

Add any antibiotics used to select the strains properly after the medium temperature is blew 37 oC.

===3. General Protocols For High-Cell-Density Media===

For 1 L media preparation

50 mM Na2HPO4.7H2O

25 mM KH2PO4 (pH 8.0-8.2)

10 mM NaCl

5 mM MgSO4

0.2 mM CaCl2

0.1% NH4Cl or 15NH4Cl

1.0% Glucose or 13C-Glucose

To prepare the media, glucose and metal solution are filtered to sterilize. The salt solution is autoclaved and the cooled solution is added by metal solution and glucose.

1D Macro

2022-07-21T20:34:16Z

Mjeppesen:

==Macro Script==
[[Image:1D_NMR_Macro.png]]

----

All the lines with a '''>''' in front are the executed lines, all the lines with a '''#''' are commented out.

The first ''three'' lines after the description are used to process the fid data. Line 1 proves to the macro that this is a proton fid.

The second line provides window functions, in the shown macro, it includes an exponential ************

The third **************

----

Next lines are the processing after the fid has been turned into a spectrum, with the first line after ensuring the file is the correct file type.

----

The last three lines are the typically changed lines. '''It is important to change the save location of the processed spectra and ASCII files.''' This

[[category:NMR_Usage|Macro]]

M9 Minimal Media

2022-07-21T20:33:26Z

Mjeppesen:

M9 Minimal Media

Per liter of sterile water:

*5X M9 salts* 200 mL
*1M MgSO4 2 mL
*20% glucose** 20 mL
*1M CaCl2 0.1 mL

5X M9 salts

Per liter:

*Na2HPO4 64 grams
*KH2PO4 15 grams
*NaCl 2.5 grams
*NH4Cl 5 grams

In this recipe, 15N ammounium chloride and 13C glucose can be used to label proteins.

Glucose cannot be sterilized in the autoclave. It must be passed through a ~0.2μm membrane to be sterilized.

[[category:Protein_Preparation]]
[[category:Cell_Culturing]]

Luria-Bertani Media

2022-07-21T20:33:14Z

Mjeppesen:

Luria-Bertani Media

Per liter:

*Tryptone (Shelf A) 10 grams
*Yeast Extract (Shelf A) 5 grams
*NaCl (Shelf A) 10 grams

Adjust to desired pH.

[[category:Protein_Preparation]]
[[category:Cell_Culturing]]

Finding a Protein Target on the NESG website

2022-07-21T20:32:59Z

Mjeppesen:

Finding a Protein Target (on the NESG website)
# Login: proteins Password: 2study
# Start by focusing your search on a particular organism: human, myco, staph…
# Begin by clicking targets labeled with “Good HSQC”. This ensures that the protein is amenable to NMR structure analysis. At this time it is also advisable to choose proteins with a MW below 20kDa.
# On the protein target page there will be a search against the PDB to see if the structure has been solved or for high similarity. Make sure the similarity is well below 50%. A higher similarity would only be good for unsolved human proteins.
# Scroll down to find the expression data. Make sure the protein is expressable and soluble. A score of 4 or 5 for each is best.
# Check the HSQC to make sure it really is “Good”. The peaks should be well resolved and there should be 1 peak per amino acid.
# Write down the plasmid ID for the protein target.

[[category:Protein_Preparation]]

Lab Notebook Guidelines

2022-07-21T20:32:38Z

Mjeppesen:

== General Rules and Regulations ==

A lab notebook is a legal document!
* All work must be thoroughly and accurately recorded
* Keep the notebook current and up to date
* Lab notebook should be a complete record of ALL of your research efforts
** Include both successful and unsuccessful experiments or data analysis
** Even seemingly trivial work done in the lab must be properly recorded
** Detailed experimental methods only need to be entered ONCE into the lab notebook
*** When repeating the experiment, just refer to the lab notebook page containing the original methods and simply note all modifications to the original protocol.
** Lab notebooks are not a personal diary
* Lab notebooks should not leave the office or lab
* Lab notebooks do not belong to you. They are the property of the university.
Everything must be neatly written in ink and legible to all readers
* Experiments and analysis should be easily reproducible from only the notebook
Maintain a Table of Contents in the front of each lab notebook

All documents and figures must be secured on a lab notebook page and within the defined page’s boarders

All reports, notes, raw data, software, and received samples should be documented and date stamped in the notebook or appendix
* All large results should be included in an appendix (software output or source code, detailed reports, processing scripts, etc.)
*A three-ring binder is an acceptable appendix.
**The binder should be labeled with Name, lab notebook number and appendix number.
**The binder should include paper inserts to provide page numbers, project titles and dates for each appendix entry.
*Location of large results in appendix should be documented in the lab notebook (i.e., page and appendix number should be cross-listed in both lab notebook and appendix)
*A reference to the location (i.e., directory path, instrument, specific computer name) of ALL raw data and digital documentation should be included in the lab notebook
*Physical description and location of ALL samples should be referenced in the lab notebook (including location (i.e, room number, freezer name, dates, name, and abbreviations).
All digital data should be backed up and archived
* Location of archived data should be noted in the lab notebook
As appropriate, a separate notebook may be used for each individual projects

Every completed lab notebook page must include a signature by the inventor, recorder, and witness
*Inventor and recorder is the person who did the work and recorded the work
*Witness is the person who read, understood, and reviewed the work
Significant unused space should be crossed out with X, initialed and dated

No pages should be left blank
*All entries should be recorded in chronological order
Outcomes, conclusions, and results of any project good or bad need to be recorded!
*Your documented and dated insights, interpretations or hypothesis are critical to supporting any patents or intellectual property.
*Also critical to establishing your ownership of your thesis work

== Lab Notebook Violations ==

*Do not tear out pages
*Do not leave blank pages between entries
*Everything should be recorded in chorological order
*Do not use white out or anything erasable
*Strike through all mistakes, do not make the mistake illegible
*Do not write sloppily, or use vague terms
**What is written in the notebook should be legible and understandable to anyone reading the notebook; and the experiments easily reproduced by reading the lab notebook
*Do not make changes to a lab notebook after it has been signed and witnessed.
**If a mistake needs to be corrected, then the change should be dated and initialed
*Do not keep loose paper consisting of notes, data or figures shoved between notebook pages
*Do not make entries into your lab notebook weeks or months after you actually completed the experiments or data analysis
**Record your experiments and data analysis as you are doing the work and obtaining the results
*Avoid spills, ripping or tearing pages, or anything that makes the lab notebook page unreadable
*Do not write long rambling entries, be succinct and precise.
**A series of numbered steps or bullet points is preferable
*Do not include confusing or uninterpretable figures or tables
**Make sure all tables and figures are properly, labeled, annotated and explained
***Axis labels with units
***All sample symbols/colors identified
***Quality factors are included (R2, Q2, p-values, etc.)
***Explain how the data was fit or modeled, the validity of the fit/model

== Completed lab notebook page should contain ==

All sections should be labeled, separated and easily distinguishable

*Title
*Purpose or Objective
*Description of Experiment (i.e., methods and materials)
**Note the name and versions of any software or instrumentation used
*Sample preparation
*Instrument description and set up
*Results
**Appendix may be added for extended results including ligand libraries, metabolite lists, software code, software output, processing scripts, peak lists, chemical shift assignments, PDB files reports, or published work
**Essentially, any result or outcome that needs to be recorded, but cannot easily fit on a single lab notebook page.
*Figures and figure legends
*Preliminary conclusions and comments
**Record any insights, thoughts or hypothesis
**Include any relevant literature citations
*Author Signature and date
*Witness Signature and date

[[category:Needs_Updating]]
[[category:General_Maintenance]]

FAST-NMR

2022-07-21T20:32:15Z

Mjeppesen:

It's comin, ya'll! This will be the page the describes the overall process of FAST-NMR and links to the protocols of the individual steps involved in FAST-NMR
[[category:FAST-NMR]]

FAST-NMR

2022-07-21T20:32:08Z

Mjeppesen:

It's comin, ya'll! This will be the page the describes the overall process of FAST-NMR and links to the protocols of the individual steps involved in FAST-NMR
[[category:FAST_NMR]]