Transformation

From 2011.igem.org

(Difference between revisions)

Latest revision as of 00:45, 29 September 2011

Materials:

Chemically competent cells

DNA plasmid ligation

SOC medium

LB+Agar plater with the appropriate antibiotic

Procedure:

1. Thaw the competent cells on ice for 5min. ( competent cells are extremely susceptible to heat, work with them ALWAYS on ice unless specified not to)

2. Meanwhile the cells are thawing out, add 2-4ul of DNA to a 2ml microtube on ice. (use molecular grade H2O for your negative control)

3. Add 50ul of the thawed out competent cells to to 2ml microtube

4. Incubate on ice for 30 min.

5. Heat shock the cells at 42°C for exactly 60 seconds and immediately return them to ice.(this heat shock time is optimized for E.coli K-12, JM109 strands)

6. Incubate 5 minutes in ice.

7. Add 200ul of room temperature SOC medium to the microtubes. (It is no longer necessary to keep the cells on ice from this step onwards)

8. Incubate for 1-2 hours at 37°C with vigorous shaking.

9. Spread 100ul of the transformants onto an agar plate with the appropriate antibiotic.

10. Incubate at 37°C "overnight" with the top side looking downwards.

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@@ Line 1: / Line 1: @@
-==Transformation of Dpn1-digested plasmid==
+'''Materials:'''
-July 2011 1pm
-Transformed contents of tubes 3a, 3b, 4a using [[Protocols:transformation|transformation]] protocol with the following changes:
+Chemically competent cells
-* 2µl of mutagenesis mix used instead of 1µl.
-* LB media instead of SOC.
-Transformanda on ice at 1305h.
+DNA plasmid ligation
+SOC medium
+LB+Agar plater with the appropriate antibiotic
+'''Procedure:'''
+. Thaw the competent cells on ice for 5min. ( ''competent cells are extremely susceptible to heat, work with them ALWAYS on ice unless specified not to'')
+. Meanwhile the cells are thawing out, add 2-4ul of DNA to a 2ml microtube on ice. (''use molecular grade H2O for your negative control'')
+. Add 50ul of the thawed out competent cells to to 2ml microtube
+. Incubate on ice for 30 min.
+. Heat shock the cells at 42°C for exactly 60 seconds and  immediately return them to ice.(''this heat shock time is optimized for E.coli K-12, JM109 strands'')
+. Incubate 5 minutes in ice.
+. Add 200ul of room temperature SOC medium to the microtubes. (''It is no longer necessary to keep the cells on ice from this step onwards'')
+. Incubate for 1-2 hours at 37°C with vigorous shaking.
+. Spread 100ul of the transformants onto an agar plate with the appropriate antibiotic.
+. Incubate at 37°C "overnight" with the top side looking downwards.
+[https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back''']