Team:Glendale CC AZ/Protocols/Transformation

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Revision as of 03:01, 23 September 2013

iGEM

Glendale Community College ArizonaGCC

Protocols

iGEM Growth Curve Assay

NaCl Growth Curve Assay

Survival Growth Assay

Alkaline Lysis Plasmid Miniprep

Restriction Digest

DNA Isolation

Bioinformatics

Ligation

Transformation

Transformation Protocol

Protocol from: http://parts.igem.org/Help:Protocols/Transformation

Materials 

  -Resuspended DNA
  -Competent cells (50ul per transformation)
  -Ice
  -2ml tube (1 per a transformation)
  -42ºC water bath
  -SOC media
  -Petri dishes with LB agar and appropriate antibiotic (2 per transformation)
  -Spreader
  -37ºC incubator
  -10pg/µl RFP Control (pSB1A3 w/ BBa_J04450)

Procedure

1. Start thawing the competent cells on ice. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.

2. Add 1 - 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.

3. Add 1 µL of the RFP Control to your control transformation.

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.

9. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.

10. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.

11. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up.

12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

13. Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.

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