This is how you make an agar plate.

This is how you make LB Broth.

Materials

• 0.5% (w/v) yeast extract
• 2% (w/v) tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 20 mM MgSO4

Per liter:

• 5 g yeast extract
• 20 g tryptone
• 0.584 g NaCl
• 0.186 g KCl
• 2.4 g MgSO4

Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4. SOB medium is also available dry premixed from Difco, 0443-17.
Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

15/10 medium

Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:

• 1.5% yeast extract
• 1% Bacto-Tryptone
• 10mM NaCl
• 2mM KCl
• 10 mM MgCl2
• 10 mM MgSO4

Estimated time: 3 hours (plus 14-18 hour incubation)

Materials

2µl resuspended DNA

Competent cells (50ul per transformation)

Ice

2ml tube

42ºC water bath

SOC or LB media

1 LB plate and 1 LB+antibiotic per transformation

Spreader

37ºC incubator

Shaker

Procedure

1. Thaw competent cells on ice.
2. Add 50µL of thawed competent cells into pre-chilled 2ml tube.
3. Add 2µL of resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Keep the competent cells on ice.
4. Close tubes and incubate the cells on ice for 30 minutes.
5. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
6. Incubate the cells on ice for 5 minutes.
7. Add 200 μl of SOC or LB media to each transformation
8. Incubate the cells at 37ºC for 2 hours at 260rpm in the shaker.
9. Label petri dishes with the appropriate antibiotic with the part number, plasmid backbone, antibiotic resistance, and date. Plate 200 µl of the transformation onto the labeled antibiotic dishes, and spread. This helps ensure that you will be able to pick out a single colony.
10. Spread remaining transformation onto labeled LB agar plate and spread, approx 50µl.
11. Incubate the plates at 37ºC for 12-18 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Controls

Competent cells on LB plate

Competent cells on LB + antibiotic plate

Competent cells + plasmid on LB plate

This is how you miniprep.

This is how you do restriction digest.

This is how you do ligation.

This is how you do gel electrophoresis.

This is how you do gel extraction.

This is the conditions we used for PCR.

This is the conditions we used for PCR cleanup.

This is the conditions we used to express luciferase.

This is the conditions we used to express chromoproteins.