LB-Agar Plates

This is how you make an agar plate.

LB Broth

This is how you make LB Broth.

SOC Media

This is how you make SOC media.

Transformation

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

Materials

2µl resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)

Competent cells (50ul per transformation)

Ice (in ice bucket/container)

2ml tube (1 per a transformation')

42ºC water bath

SOC media

1 small LB plate and 1 large LB+antibiotic per transformation

Spreader

37ºC incubator

Procedure

1. Start thawing the competent cells on ice.
2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
3. Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to 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 media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation
8. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
9. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. 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.

Miniprep

This is how you miniprep.

Restriction Digest

This is how you do restriction digest.

Ligation

This is how you do ligation.

Gel Electrophoresis

This is how you do gel electrophoresis.

Gel Extraction

This is how you do gel extraction.

PCR

This is the conditions we used for PCR.

PCR Cleanup

This is the conditions we used for PCR cleanup.

Luciferase

This is the conditions we used to express luciferase.

Chromoproteins

This is the conditions we used to express chromoproteins.