Plasmid Purification and Transformation Protocol

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

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

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