Main Page

General microbiology protocols

Transformation

1. Turn on heat block to 42 degrees. 2. Turn on shaking water bath to 37 degrees. 3. Spray a beaker with ethanol, go get ice.

4. Label 3 1.5mL microfuge tubes with pUC18, pUC18-E, water 1. Get 6 tubes of blue 100uL DH5alpha cells from -80 freezer. (Each tube has 100uL, get 6 you have 600. 250 for the ligated and plasmid condition, then 100 leftover for the negative control water).

5. Transfer 250uL of bacterial cells to each of your ligated and empty plasmid microfuge tubes. Transfer 100uL to the water tube. Spin for 5 minutes at 10,000 rpm. This will cause the bacterial cells to collect in a pellet at the bottom of your tube.

6. Remove the supernatant without disrupting the bacterial pellets. Add 25 µL of ice cold 1x TSS buffer/tube (10uL for the water tube) and resuspend the cells by flicking the tube gently, then place the tubes on ice. [TSS buffer is LB with detergents and salts which will permeabilize cell membranes, allowing the plasmid to enter the bacterial cells].

7. Add 10 (25ng) µL of plasmid solution to the ligated plasmid tubes. Add 0.23uL (for 25ng) of pUC18-1 or (0.4 for pUC18-2), to the empty vector tubes of cells. Add 10uL of sterile water to the 7th tubes for a negative control. Place back on ice. As you add these small volumes, mix gently with tip of your pipette.

8. Allow the cells to incubate for 40 minutes on ice. Do not mix the tubes during this incubation period.

9. After 40 mins, carry the ice bucket to a heat block pre-heated to 42 degrees and heat shock the cells for exactly 45 seconds, then immediately return the tubes to ice for 2 minutes. Add 250 ml of room temperature LB to each of your tubes.

10. Incubate your microfuge tubes with cells in ‘floaties’ for one hour in the shaking water bath, set to 37°C.

11. After 60 minutes, plate as follows:

Getting started with MediaWiki

Consult the User's Guide for information on using the wiki software.

• Configuration settings list
• MediaWiki FAQ
• MediaWiki release mailing list
• Localise MediaWiki for your language
• Learn how to combat spam on your wiki