Powers Wiki - User contributions [en]

Filling a Magnet with Nitrogen

2021-01-07T19:24:44Z

Fatema:

Filling a Magnet with Nitrogen**

# Find a rubber transfer line, two additional short pieces of rubber, and a glove.
# Wheel the low pressure dewar up to the magnet to be filled. Position the liquid transfer valve on the dewar near (4 ft away) the nitrogen fill port on the magnet. The fill port will be labeled.
# Attach the transfer line to the liquid valve on dewar.
# Remove the blue cover from the liquid nitrogen fill port.
# Slide the transfer line on the end of the fill port.
# Remove the covers to the vent port(s) and replace with short pieces of rubber tubing. This ensures the excess nitrogen is not sprayed toward the ceiling. The 600 and 700 have one vent port and the 400 and 300 have two vent ports.
# With one hand shape the transfer line so that there are no kinks in the tubing.
# With the other hand slowly open the liquid valve. Transfer nitrogen slowly at this point.
# After the transfer line freezes in place you can open the liquid valve more. The line should have a slight shake when the speed is appropriate.
# Replace the transfer line and rubber pieces with the port covers when the fill is complete. The fill will be complete when liquid nitrogen starts spitting out of the vent ports.
# Fill out the log book.

Using and Maintaining pH Meter

2021-01-07T02:06:17Z

Fatema: Created page with "Be careful with the probe. It is very fragile. Calibrate the instrument with ph 4,7,10 solutions prior to your usage, each time."

Be careful with the probe. It is very fragile.
Calibrate the instrument with ph 4,7,10 solutions prior to your usage, each time.

Category:Protocols

2021-01-07T01:47:18Z

Fatema:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Requesting Balance Calibration]]
*[[Requesting Pipette Calibration]]
*[[Using the UV-Vis]]
*[[Using and Maintaining pH Meter]]
*[[-80 Freezer Storage and Maintenance]]
*[[Freeze Dryer Maintenance]]
*[[Lab Notebook Guidelines]]
*[[Sample Barcoding]]

''Protein Preparation''
*[[Buffer Exchange and Solution Concentration]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]

''Data Collection''
*[[Gap Sampling]]
*[[Water Suppression with presaturation pulses (zgpr/zgcppr]]
*[[Non-uniform Sampling]]
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]

''Data Processing and Analysis''
*[[Analysis of 1D Line-Broadening Screen]]
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
*[[Non-Uniform Sampling]]
*[[NMRFAM-SPARKY Guide]]
*[[NMRFAM-SPARKY: Automated Peak Assignment]]
*[[2D NMR Processing in Linux and Windows]]
*[[2D Metabolomics NMRPipe Processing]]
*[[1D NMR Processing in Linux and Windows Example Script]]
*[[Sample Collection and Processing for Protein Backbone Assignments]]
*[[Example Scripts for NMRPipe Processing]]

''Miscellaneous''
*[[Agarose Gel]]
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Cell Culturing
*[[Cell Culturing Dr. Franco Lab]]

''Metabolomics''
*[[Metabolite Extraction]]
*[[MetPa for metabolomics]]
*[[Making Heatmaps]]
*[[One way ANOVA in R]]
*[[P-Value adjustment for multiple comparisons]]
*[[PCA-Utils]]
*[[NMR Tube Deep Cleaning]]
*[[Noise Removal for PCA]]
*[[Weighted Linear Least Squares]]
*[[Serum Preparation for 1D NMR]]
*[[Whole Blood Preparation for 1D NMR]]
*[[Urine Preparation for 1D NMR]]
*[[Cell Culture Preparation for 1D NMR]]
*[[LC-MS metabolomics guide-CIBC collaborations]]

''Safety and Inspections''
*[[Lab Responsibilities]]
*[[Inspection Checklist]]
*[[Safety Contacts, Resources]]

''Demos''
*[[Peroxide Clock]]
*[[Traffic Light Reaction]]
*[[Orange Juice Clock]]
*[[Gummy Bear Freeze]]
*[[Lava Lamps]]
*[[Batteries]]
*[[Rocket Launcher]]
*[[Women in Science: Checklist]]
*[[Maxey Day: Checklist]]

Metabolite Extraction

2021-01-07T01:39:43Z

Fatema: Created page with "==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..."

==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:Protocols

2021-01-07T01:39:24Z

Fatema:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Requesting Balance Calibration]]
*[[Requesting Pipette Calibration]]
*[[Using the UV-Vis]]
*[[Using and Maintaining pH Meter]]
*[[-80 Freezer Storage and Maintenance]]
*[[Freeze Dryer Maintenance]]
*[[Lab Notebook Guidelines]]
*[[Sample Barcoding]]

''Protein Preparation''
*[[Buffer Exchange and Solution Concentration]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]

''Data Collection''
*[[Gap Sampling]]
*[[Water Suppression with presaturation pulses (zgpr/zgcppr]]
*[[Non-uniform Sampling]]
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]

''Data Processing and Analysis''
*[[Analysis of 1D Line-Broadening Screen]]
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
*[[Non-Uniform Sampling]]
*[[NMRFAM-SPARKY Guide]]
*[[NMRFAM-SPARKY: Automated Peak Assignment]]
*[[2D NMR Processing in Linux and Windows]]
*[[1D NMR Processing in Linux and Windows Example Script]]
*[[Sample Collection and Processing for Protein Backbone Assignments]]
*[[Example Scripts for NMRPipe Processing]]

''Miscellaneous''
*[[Agarose Gel]]
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Cell Culturing
*[[Cell Culturing Dr. Franco Lab]]

''Metabolomics''
*[[Metabolite Extraction]]
*[[MetPa for metabolomics]]
*[[Making Heatmaps]]
*[[One way ANOVA in R]]
*[[P-Value adjustment for multiple comparisons]]
*[[PCA-Utils]]
*[[NMR Tube Deep Cleaning]]
*[[Noise Removal for PCA]]
*[[Weighted Linear Least Squares]]
*[[Serum Preparation for 1D NMR]]
*[[Whole Blood Preparation for 1D NMR]]
*[[Urine Preparation for 1D NMR]]
*[[Cell Culture Preparation for 1D NMR]]
*[[LC-MS metabolomics guide-CIBC collaborations]]

''Safety and Inspections''
*[[Lab Responsibilities]]
*[[Inspection Checklist]]
*[[Safety Contacts, Resources]]

''Demos''
*[[Peroxide Clock]]
*[[Traffic Light Reaction]]
*[[Orange Juice Clock]]
*[[Gummy Bear Freeze]]
*[[Lava Lamps]]
*[[Batteries]]
*[[Rocket Launcher]]
*[[Women in Science: Checklist]]
*[[Maxey Day: Checklist]]

Requesting Balance Calibration

2021-01-07T01:09:39Z

Fatema: Created page with "Contact with department service-stock room."

Contact with department service-stock room.

Requesting Pipette Calibration

2021-01-07T01:01:56Z

Fatema:

Contact with Rene/department service.
It repeats annually.

Requesting Pipette Calibration

2021-01-07T01:01:20Z

Fatema: Created page with "Contact with Rene/department service"

Contact with Rene/department service

Main Page

2021-01-06T19:57:45Z

Fatema:

'''Welcome to the BioNMR Wiki Page.'''

This wiki exists to provide better access to lab protocols for the Powers lab. It is not a replacement for lab notebooks. Feel free to begin documenting new lab protocols or procedures here, or even moving old protocols onto this wiki.

[[Special:Categories|View All Categories]]

Commonly used categories:

* [[:Category:Protocols|Protocols]]
* [[:Category:Bioscreen|Bioscreen]]
* [[:Category:FAST-NMR|FAST-NMR]]
* [[:Category:Molecular Docking|Molecular Docking]]
* [[:Category:Metabolomics|Metabolomics]]
* [[:Category:Maxey Demos|Maxey Demos]]
* [[:Category:Protein expression|Protein expression]]
* [[:Category:SMACMS|SMACMS]]
* [[:Category:Lab Safety | Lab Safety]]
* [[:Category:Cell culture | Cell culture and cell passages]]
* [[:Protein Software]]

And, very likely, an unpopular category:
* [[:Category:Sysadmin | System Administration]]

Main Page

2021-01-06T19:57:17Z

Fatema:

'''Welcome to the BioNMR Wiki Page.'''

This wiki exists to provide better access to lab protocols for the Powers lab. It is not a replacement for lab notebooks. Feel free to begin documenting new lab protocols or procedures here, or even moving old protocols onto this wiki.

[[Special:Categories|View All Categories]]

Commonly used categories:

* [[:Category:Protocols|Protocols]]
* [[:Category:Bioscreen|Bioscreen]]
* [[:Category:FAST-NMR|FAST-NMR]]
* [[:Category:Molecular Docking|Molecular Docking]]
* [[:Category:Metabolomics|Metabolomics]]
* [[:Category:Maxey Demos|Maxey Demos]]
* [[:Category:Protein expression|Protein expression]]
* [[:Category:SMACMS|SMACMS]]
* [[:Category:Lab Safety | Lab Safety]]
* [[:Category:Cell culture | Culture and passage cells]]
* [[:Protein Software]]

And, very likely, an unpopular category:
* [[:Category:Sysadmin | System Administration]]

LC-MS metabolomics guide-CIBC collaborations

2020-06-22T01:08:53Z

Fatema:

Powers’ Laboratory Guide for Metabolomics Experiments (written by Aline De Lima Liete, Amith Maroli editted by Isin Tuna Sakallioglu) This guide provides a good practical foundation to guide our users through basics steps involved in metabolomic analysis by liquid chromatography mass spectrometry, gas chromatography mass spectrometry. These guidelines can be applied to different types of samples to improve the quality of the results.

Please reach out Isin Tuna Sakallioglu from isin-tuna.sakallioglu@huskers.unl.edu to see the complete PDF file with figures. Further questions while following the protocols could be asked to Dr. Liete and Dr. Maroli. [https://ncibc.unl.edu/guide-metabolomics-experiments] this LINK has similar information.

MS analysis are very expensive labor we should let the colloborators know about the details of the cost before starting the project! You may see chapter I for the details. NOTE: IT IS ESSENTAIL TO SEND AN EXCEL SPREADSHEET THAT HAS DETAILS OF ALL SAMPLES AND DETAILED TOTAL COST OF THE PROJECT TO THE COLLOBORATOR. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples and to avoid any kind of financial conflict with the colloborator.

You MUST completely AVOID! STABILIZERS: DO NOT USE any glycerol, PEG or related stabilizers. They are easy to ionize, especially PEG, and if they are present, that is all you will see! You MUST AVOID DETERGENT for the vials you are using for mass spectrometry. COLORED TUBES AND TIPS: DO NOT USE IN ANY STEP. These materials often contain residual quantities of dyes that can be release into your sample. We provide some options "mass spec friendly" list. DO NOT SEAL TUBES WITH PARAFILM. They are a rich source of PEG contamination.

A COUPLE OF VERY IMPORTANT THINGS TO AVOID OTHER CONTAMINANTS 1. Any sample manipulation should be done in a BSC, laminar flow hood, or by using mask and disposable surgical cap. 2. Always wear nitrile (not latex) gloves. 3. Wear a lab coat and make sure there is no gap between your coat sleeve and the gloves (lab tape and rubber bands work well). 4. All the bottles and containers that are purchased for MS metabolomics should be dedicated for MS metabolomics use only. Do not wash these with detergent. Do not share with others in your lab for non-MS purposes.

RECOMMENDED REAGENTS AND MATERIALS Vendor Cat# Item Brand FisherSci PI85188 Acetonitrile LC/MS Grade thermo Chemical Thermo-Pierce FisherSci 14-432-22 Falcon 50mL Conical Centrifuge Tubes Fischer FisherSci 14-959-53A Falcon™ 15mL Conical Centrifuge Tubes Fischer FisherSci 19-177- 520 Gloves, Nitrile; Microflex XCEED; Latex free Xceed FisherSci 10-320-734 Low-Binding Rack Pipet Tips 10 uL Corning FisherSci 02-717-150 Low-Binding Rack Pipet Tips 1000 uL Fischer FisherSci 10-320-735 Low-Binding Rack Pipet Tips 200 uL Corning FisherSci A456-212 Methanol, Optima™ LC/MS Grade, Fisher Chemical Fischer FisherSci 13 688 654 Pipet Tips 1 to 10mL Fischer FisherSci 60-001-35 PROPANOL CHRMSLV LC-M 2.5LT Honeywell Riedel-de Haen FisherSci 02-681-320 Microcentrifuge Siliconized Low-retention 1.5 mL Fischer FisherSci 02-681-321 Microcentrifuge Siliconized Low-retention 2.0 mL Fischer FisherSci 600-30-79 Water with 0.1% Formic Acid, LC-MS and HPLC Honeywell Burdick &Jackson

SOLUTIONS All solutions must be freshly prepared. Make sure you have enough solution (mobile phase and sample reconstitute) for all the samples, prior to your run. All reagents must be at least HPLC grade for MS experiments. For more information consult the list of recommended material. All these protocols provided are suggestions and provide good starting point. The results can vary according to sample. You can optimize the protocols for your experiment.

Extraction solution A = 100% Methanol containing one IS for positive mode and one for negative mode. Extraction solution B = 0.1% Formic Acid in Water (LC-MS grade) containing one IS for positive mode and one for negative mode. Extraction solution C = Methanol/Water (1:1) containing one IS for positive mode and one for negative mode. Extraction solution D = Dichloromethane/methanol (3:1) containing one IS for positive mode and one for negative mode. Extraction solution E = Chloroform/Methanol/Water (1:3:1 ratio) containing one IS for positive mode and one for negative mode. Extraction solution F = 80% Methanol containing one IS for positive mode and one for negative mode...

Internal Standards (could be stable isotope labelled) Mixture Components Positive mode Negative mode Expected mass ug/mL Expected mass ug/mL Caffeine 195.0882 1.5 Not applicable - Sulfadimethoxine (SDM) 311.0814 1 309.0658 1 Reserpine 609.2812 0.6 Not applicable - Leucine-Enkephalin (Leu-Enk) 556.2771 2.5 554.2615 2.5 4-Nitrobenzoic acid (NTA) Not applicable - 166.0146 10 Sulfaguanidine (SG) 215.0603 5 213.0446 5

Sample type Caffeine SDM Reserpine Leu-Enk NTA SG Human + (see note) + + (see note) - + + Rodent + (see note) + (see note) + (see note) - + + (see note) Mammalian cells + + + - + + Microbial cells + + + + + +

Important information Study design Definition of technical and biological replicates depends on the model. For omics between 4-6 replicates is a good starting point for a pilot study. Depending on your study subject (animals, cells etc.) your replicate number should change. Cultured cells n=5-10, for animal models=7-12, for plant models n=7-12, for controlled clinical trials n=12-25, uncontrolled clinical trials n=50-100, epidemiology studies n=100-1000. (these numbers are taken from “Waters Metabolomics workshop study design data analysis”, they might vary)

Quality Control (QC) The purpose of QCs is to monitor the performance of metabolomics workflows (extraction and acquisition). You should prepare QCs before extraction by mixing equal volumes from each sample and continue same protocol of extraction with all your samples and QC, together. Preparation of the QCs should follow the same protocol performed for samples including number of freeze-thaw cycles. If it is not possible to create a pooled QC sample due to limited sample amounts commercially available QC sample may be used (ex: human serum)

Blank You should do the same extraction protocol with only solutions and tubes you have been using for the other samples. That should not contain any sample or QC. This will eliminate false discoveries.

Internal standard(s) Added in first step of sample extraction, generally at the extraction solution. Please select your internal standard wisely prior to your extraction, it should be exogenous and not present in your subject. Prefer labelled sample.

Randomization Randomization of sample preparation order to avoid systematic bias. In an ‘omics’ experiment, a batch is defined as a set of samples that have been extracted as well as measured in one uninterrupted sequence. Most often, batch effects in large untargeted metabolomics experiments are almost unavoidable and hence the goal of batch correction is to remove these between-batch and within-batch effects. Accounting for these batch effects ensures that the measurements across all batches are directly comparable. One of the key steps for mitigating systematic bias is ensuring randomization of samples and sample run order. Ideally, randomization of samples should be implemented right at the start of the experiment (from sample handling, processing) till the conclusion of the run on the instrument. The key goal of sample randomization is to achieve a COMPLETELY RANDOMIZED AND BALANCED sample set.

Chapter I – Sample Label and Excel Spreadsheet if you are doing Colloboration MS analysis are very expensive labor we should let the colloborators know about the details of the cost before starting the project! - All samples MUST to be labelled or bar-coded and an Excel sheet should be provided to the colloborators before MS analysis. - Label must be made on both, lids and sides of the tubes, with water resistant markers or bar code tags. - The labelling system should be unique and contain PI initials (two letters), in charge student initials (two letters) and numbered form 001-1000…. - All the tubes MUST to be in a Microtube Storage Boxes - All Storage boxes MUST have a tag in the lid (Figure 2) containing all important information about samples. Ex: PI: Robert Powers, Student: John Doe, Samples 1-10 Label as F RPJD001, RPJD002 ….RPJD010

Project: Yeast metabolome PI: Robert Powers Student: John Doe Sample type: Yeast cell pellet Collected Date: 01/31/2019 Delivered Date: Analysis: MS untargeted metabolomics Project cost: Comment:Samples defrosted 1x NOTE: IT IS ESSENTAIL TO SEND AN EXCEL SPREADSHEET THAT HAS DETAILS OF ALL SAMPLES AND DETAILED TOTAL COST OF THE PROJECT TO THE COLLOBORATOR. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples and to avoid any kind of financial conflict with the colloborator. (Reach out Aline De Lima Liete a.lima@unl.edu for the details of the excel spreadsheet).

Chapter II – Sample collection, storage and transport Please reach out either Isin isin-tuna.sakallioglu@huskers.unl.edu or Aline De Lima Liete a.lima@unl.edu for this chapter.

Chapter III – Metabolites extraction Please reach out either Isin isin-tuna.sakallioglu@huskers.unl.edu or Aline De Lima Liete a.lima@unl.edu for this chapter.

Appendix Please reach out Amith Maroli amith.maroli@unl.edu for the following informations: I. Randomization II. Bar-coding protocol (very important step in storage of the samples, learn it well!)

LC-MS metabolomics guide-CIBC collaborations

2020-06-22T00:47:19Z

Fatema: Created page with "Powers’ Laboratory Guide for Metabolomics Experiments (written by Aline De Lima Liete, Amith Maroli editted by Isin Tuna Sakallioglu) This guide provides a good practical fo..."

Powers’ Laboratory Guide for Metabolomics Experiments (written by Aline De Lima Liete, Amith Maroli editted by Isin Tuna Sakallioglu) This guide provides a good practical foundation to guide our users through basics steps involved in metabolomic analysis by liquid chromatography mass spectrometry, gas chromatography mass spectrometry. These guidelines can be applied to different types of samples to improve the quality of the results.

Please reach out Isin Tuna Sakallioglu from isin-tuna.sakallioglu@huskers.unl.edu to see the complete PDF file with figures. Further questions while following the protocols could be asked to Dr. Liete and Dr. Maroli. [1] this LINK has similar information.

MS analysis are very expensive labor we should let the colloborators know about the details of the cost before starting the project! You may see chapter I for the details. NOTE: IT IS ESSENTAIL TO SEND AN EXCEL SPREADSHEET THAT HAS DETAILS OF ALL SAMPLES AND DETAILED TOTAL COST OF THE PROJECT TO THE COLLOBORATOR. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples and to avoid any kind of financial conflict with the colloborator.

You MUST completely AVOID! STABILIZERS: DO NOT USE any glycerol, PEG or related stabilizers. They are easy to ionize, especially PEG, and if they are present, that is all you will see! You MUST AVOID DETERGENT for the vials you are using for mass spectrometry. COLORED TUBES AND TIPS: DO NOT USE IN ANY STEP. These materials often contain residual quantities of dyes that can be release into your sample. We provide some options "mass spec friendly" list. DO NOT SEAL TUBES WITH PARAFILM. They are a rich source of PEG contamination.

A COUPLE OF VERY IMPORTANT THINGS TO AVOID OTHER CONTAMINANTS 1. Any sample manipulation should be done in a BSC, laminar flow hood, or by using mask and disposable surgical cap. 2. Always wear nitrile (not latex) gloves. 3. Wear a lab coat and make sure there is no gap between your coat sleeve and the gloves (lab tape and rubber bands work well). 4. All the bottles and containers that are purchased for MS metabolomics should be dedicated for MS metabolomics use only. Do not wash these with detergent. Do not share with others in your lab for non-MS purposes.

RECOMMENDED REAGENTS AND MATERIALS Vendor Cat# Item Brand FisherSci PI85188 Acetonitrile LC/MS Grade thermo Chemical Thermo-Pierce FisherSci 14-432-22 Falcon 50mL Conical Centrifuge Tubes Fischer FisherSci 14-959-53A Falcon™ 15mL Conical Centrifuge Tubes Fischer FisherSci 19-177- 520 Gloves, Nitrile; Microflex XCEED; Latex free Xceed FisherSci 10-320-734 Low-Binding Rack Pipet Tips 10 uL Corning FisherSci 02-717-150 Low-Binding Rack Pipet Tips 1000 uL Fischer FisherSci 10-320-735 Low-Binding Rack Pipet Tips 200 uL Corning FisherSci A456-212 Methanol, Optima™ LC/MS Grade, Fisher Chemical Fischer FisherSci 13 688 654 Pipet Tips 1 to 10mL Fischer FisherSci 60-001-35 PROPANOL CHRMSLV LC-M 2.5LT Honeywell Riedel-de Haen FisherSci 02-681-320 Microcentrifuge Siliconized Low-retention 1.5 mL Fischer FisherSci 02-681-321 Microcentrifuge Siliconized Low-retention 2.0 mL Fischer FisherSci 600-30-79 Water with 0.1% Formic Acid, LC-MS and HPLC Honeywell Burdick &Jackson

SOLUTIONS All solutions must be freshly prepared. Make sure you have enough solution (mobile phase and sample reconstitute) for all the samples, prior to your run. All reagents must be at least HPLC grade for MS experiments. For more information consult the list of recommended material. All these protocols provided are suggestions and provide good starting point. The results can vary according to sample. You can optimize the protocols for your experiment.

Extraction solution A = 100% Methanol containing one IS for positive mode and one for negative mode. Extraction solution B = 0.1% Formic Acid in Water (LC-MS grade) containing one IS for positive mode and one for negative mode. Extraction solution C = Methanol/Water (1:1) containing one IS for positive mode and one for negative mode. Extraction solution D = Dichloromethane/methanol (3:1) containing one IS for positive mode and one for negative mode. Extraction solution E = Chloroform/Methanol/Water (1:3:1 ratio) containing one IS for positive mode and one for negative mode. Extraction solution F = 80% Methanol containing one IS for positive mode and one for negative mode...

Internal Standards (could be stable isotope labelled) Mixture Components Positive mode Negative mode Expected mass ug/mL Expected mass ug/mL Caffeine 195.0882 1.5 Not applicable - Sulfadimethoxine (SDM) 311.0814 1 309.0658 1 Reserpine 609.2812 0.6 Not applicable - Leucine-Enkephalin (Leu-Enk) 556.2771 2.5 554.2615 2.5 4-Nitrobenzoic acid (NTA) Not applicable - 166.0146 10 Sulfaguanidine (SG) 215.0603 5 213.0446 5

Sample type Caffeine SDM Reserpine Leu-Enk NTA SG Human + (see note) + + (see note) - + + Rodent + (see note) + (see note) + (see note) - + + (see note) Mammalian cells + + + - + + Microbial cells + + + + + +

Important information Study design Definition of technical and biological replicates depends on the model. For omics between 4-6 replicates is a good starting point for a pilot study. Depending on your study subject (animals, cells etc.) your replicate number should change. Cultured cells n=5-10, for animal models=7-12, for plant models n=7-12, for controlled clinical trials n=12-25, uncontrolled clinical trials n=50-100, epidemiology studies n=100-1000. (these numbers are taken from “Waters Metabolomics workshop study design data analysis”, they might vary)

Quality Control (QC) The purpose of QCs is to monitor the performance of metabolomics workflows (extraction and acquisition). You should prepare QCs before extraction by mixing equal volumes from each sample and continue same protocol of extraction with all your samples and QC, together. Preparation of the QCs should follow the same protocol performed for samples including number of freeze-thaw cycles. If it is not possible to create a pooled QC sample due to limited sample amounts commercially available QC sample may be used (ex: human serum)

Blank You should do the same extraction protocol with only solutions and tubes you have been using for the other samples. That should not contain any sample or QC. This will eliminate false discoveries.

Internal standard(s) Added in first step of sample extraction, generally at the extraction solution. Please select your internal standard wisely prior to your extraction, it should be exogenous and not present in your subject. Prefer labelled sample.

Randomization Randomization of sample preparation order to avoid systematic bias. In an ‘omics’ experiment, a batch is defined as a set of samples that have been extracted as well as measured in one uninterrupted sequence. Most often, batch effects in large untargeted metabolomics experiments are almost unavoidable and hence the goal of batch correction is to remove these between-batch and within-batch effects. Accounting for these batch effects ensures that the measurements across all batches are directly comparable. One of the key steps for mitigating systematic bias is ensuring randomization of samples and sample run order. Ideally, randomization of samples should be implemented right at the start of the experiment (from sample handling, processing) till the conclusion of the run on the instrument. The key goal of sample randomization is to achieve a COMPLETELY RANDOMIZED AND BALANCED sample set.

Chapter I – Sample Label and Excel Spreadsheet if you are doing Colloboration MS analysis are very expensive labor we should let the colloborators know about the details of the cost before starting the project! - All samples MUST to be labelled or bar-coded and an Excel sheet should be provided to the colloborators before MS analysis. - Label must be made on both, lids and sides of the tubes, with water resistant markers or bar code tags. - The labelling system should be unique and contain PI initials (two letters), in charge student initials (two letters) and numbered form 001-1000…. - All the tubes MUST to be in a Microtube Storage Boxes - All Storage boxes MUST have a tag in the lid (Figure 2) containing all important information about samples. Ex: PI: Robert Powers, Student: John Doe, Samples 1-10 Label as F RPJD001, RPJD002 ….RPJD010

Project: Yeast metabolome PI: Robert Powers Student: John Doe Sample type: Yeast cell pellet Collected Date: 01/31/2019 Delivered Date: Analysis: MS untargeted metabolomics Project cost: Comment:Samples defrosted 1x NOTE: IT IS ESSENTAIL TO SEND AN EXCEL SPREADSHEET THAT HAS DETAILS OF ALL SAMPLES AND DETAILED TOTAL COST OF THE PROJECT TO THE COLLOBORATOR. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples and to avoid any kind of financial conflict with the colloborator. (Reach out Aline De Lima Liete a.lima@unl.edu for the details of the excel spreadsheet).

Chapter II – Sample collection, storage and transport Please reach out either Isin isin-tuna.sakallioglu@huskers.unl.edu or Aline De Lima Liete a.lima@unl.edu for this chapter.

Chapter III – Metabolites extraction Please reach out either Isin isin-tuna.sakallioglu@huskers.unl.edu or Aline De Lima Liete a.lima@unl.edu for this chapter.

Appendix Please reach out Amith Maroli amith.maroli@unl.edu for the following informations: I. Randomization II. Bar-coding protocol (very important step in storage of the samples, learn it well!)

Category:Protocols

2020-06-22T00:44:57Z

Fatema:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Requesting Balance Calibration]]
*[[Requesting Pipette Calibration]]
*[[Using the UV-Vis]]
*[[Using and Maintaining pH Meter]]
*[[-80 Freezer Storage and Maintenance]]
*[[Freeze Dryer Maintenance]]
*[[Lab Notebook Guidelines]]
*[[Sample Barcoding]]

''Protein Preparation''
*[[Buffer Exchange and Solution Concentration]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]

''Data Collection''
*[[Gap Sampling]]
*[[Water Suppression with presaturation pulses (zgpr/zgcppr]]
*[[Non-uniform Sampling]]
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]

''Data Processing and Analysis''
*[[Analysis of 1D Line-Broadening Screen]]
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
*[[Non-Uniform Sampling]]
*[[NMRFAM-SPARKY Guide]]
*[[NMRFAM-SPARKY: Automated Peak Assignment]]
*[[NMR Processing in Linux and Windows]]
*[[Sample Collection and Processing for Protein Backbone Assignments]]
*[[Example Scripts for NMRPipe Processing]]

''Miscellaneous''
*[[Agarose Gel]]
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Cell Culturing
*[[Cell Culturing Dr. Franco Lab]]

''Metabolomics''
*[[MetPa for metabolomics]]
*[[Making Heatmaps]]
*[[Metabolite Extraction]]
*[[One way ANOVA in R]]
*[[P-Value adjustment for multiple comparisons]]
*[[PCA-Utils]]
*[[NMR Tube Deep Cleaning]]
*[[Noise Removal for PCA]]
*[[Weighted Linear Least Squares]]
*[[Serum Preparation for 1D NMR]]
*[[Urine Preparation for 1D NMR]]
*[[LC-MS metabolomics guide-CIBC collaborations]]

''Safety and Inspections''
*[[Lab Responsibilities]]
*[[Inspection Checklist]]
*[[Safety Contacts, Resources]]

''Demos''
*[[Peroxide Clock]]
*[[Traffic Light Reaction]]
*[[Orange Juice Clock]]
*[[Gummy Bear Freeze]]
*[[Batteries]]
*[[Rocket Launcher]]
*[[Women in Science: Checklist]]
*[[Maxey Day: Checklist]]

Category:Protocols

2020-06-21T23:51:14Z

Fatema:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Requesting Balance Calibration]]
*[[Requesting Pipette Calibration]]
*[[Using the UV-Vis]]
*[[Using and Maintaining pH Meter]]
*[[-80 Freezer Storage and Maintenance]]
*[[Freeze Dryer Maintenance]]
*[[Lab Notebook Guidelines]]
*[[Sample Barcoding]]

''Protein Preparation''
*[[Buffer Exchange and Solution Concentration]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]

''Data Collection''
*[[Gap Sampling]]
*[[Water Suppression with presaturation pulses (zgpr/zgcppr]]
*[[Non-uniform Sampling]]
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]

''Data Processing and Analysis''
*[[Analysis of 1D Line-Broadening Screen]]
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
*[[Non-Uniform Sampling]]
*[[NMRFAM-SPARKY Guide]]
*[[NMRFAM-SPARKY: Automated Peak Assignment]]
*[[NMR Processing in Linux and Windows]]
*[[Sample Collection and Processing for Protein Backbone Assignments]]
*[[Example Scripts for NMRPipe Processing]]

''Miscellaneous''
*[[Agarose Gel]]
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Cell Culturing
*[[Cell Culturing Dr. Franco Lab]]

''Metabolomics''
*[[MetPa for metabolomics]]
*[[Making Heatmaps]]
*[[Metabolite Extraction]]
*[[One way ANOVA in R]]
*[[P-Value adjustment for multiple comparisons]]
*[[PCA-Utils]]
*[[NMR Tube Deep Cleaning]]
*[[Noise Removal for PCA]]
*[[Weighted Linear Least Squares]]
*[[Serum Preparation for 1D NMR]]
*[[Urine Preparation for 1D NMR]]
*[[LC-MS metabolomics guide-CIBC users]]

''Safety and Inspections''
*[[Lab Responsibilities]]
*[[Inspection Checklist]]
*[[Safety Contacts, Resources]]

''Demos''
*[[Peroxide Clock]]
*[[Traffic Light Reaction]]
*[[Orange Juice Clock]]
*[[Gummy Bear Freeze]]
*[[Batteries]]
*[[Rocket Launcher]]
*[[Women in Science: Checklist]]
*[[Maxey Day: Checklist]]

Category:Protocols

2016-12-20T23:50:17Z

Fatema: Blanked the page

Category:Protocols

2016-12-20T23:49:24Z

Fatema:

Creating a E.Coli Stock Culture

1. Make LB media as per the protocol titled Media Preparation

2. Transfer 25mL of LB media in to a sterile (autoclave) 250 mL erlenmeyer flask using a sterile Falcon Transfer Pipet

3. Inoculate the flask with enough stock culture(Glycerol stock obtained from a lab mate/purchased) to create a final OD600 of 0.025

4. Make note to have 1:10 ratio of liquid to air in your inoculated flask for aerobic growth of E.coli

5. Incubate the flask at 37C and a shaker speed of 100-150 rpm

6. Over every 30 min take an OD600 reading to track the growth of the E.coli cells in your flask

7. When the cells hit Stationary phase (OD600 greater than or equal to 10 in 1ml of culture) the culture is ready to make stock tubes for storage

8. At this point, take 1ml of culture and add 0.5ml of glycerol in a 2ml sterile tube, create 10-15 of these 2ml glycerol stock tubes

9. Store the tubes with appropriate labeling in the -80C freezer for until you need to use them again.

Category:Protocols

2016-12-20T23:39:13Z

Fatema:

Creating a E.Coli Stock Culture