A Brief Description of What We Will Do

From Hackteria Wiki
Revision as of 15:42, 16 June 2009 by (talk)
Jump to: navigation, search

Step 1 - Taking dry DNA from wells

1. Making sure the plates are properly oriented, punch a hole through the foil cover using a pipette - into the well you want the DNA from. Don't remove foil cover since it might pose the threat of cross-contamination between wells.

2. Add 15uL of deionized water - diH2O

Step 2 - Transforming competent cells. Competent cells are those cells which have the ability to take up extracellular/naked DNA from its environment.

To transform competent cells, we require resuspended DNA and some already competent cells.

1. Start thawing the competent cells on crushed ice.

2. Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA (i.e. DNA taken from the well) to the labelled tubes. Make sure to keep the competent cells on ice.

3. Incubate the cells on ice for 30 minutes.

4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.

5. Incubate the cells on ice for 5 minutes.

6. Add 200 μl of LB broth (make sure that the broth does not contain antibiotics and is not contaminated)

7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency.

8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.

9. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.

Step 3 - Pick a single colony.

Step 4 - Inoculate broth with Ampicillin-R (an antibiotic) and let it grow for 18 hours.

Step 5 - Use the resulting culture for mini-prep. - a process used to purify plasmids and yields clean, usable DNA.