Transformation Protocol Using Heat Shock

(Difference between revisions)

Revision as of 08:38, 22 August 2012

1) Take competent E.coli cells from –80°C freezer.

2) Turn on water bath to 42°C.

3) Put 100 ul of competent cells in an Eppendorf tube.

4) Keep tubes on ice.

5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 min. to thaw competent cells.

6) Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.

7) Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.

8) Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C.

9) Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.

10) Pick colonies about 12-16 hours later.

@@ Line 3: / Line 3: @@
 ) Turn on water bath to 42°C.
-) Put competent cells in a 1.5 ml tube (Eppendorf or similar). For transforming a DNA construct, use 50 ul of competent cells. For transforming a ligation, use 100 ul of competent cells. You may need more or less cells, depending how competent they are.
+) Put 100 ul of competent cells in an Eppendorf tube.
 ) Keep tubes on ice.
@@ Line 13: / Line 13: @@
 ) Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
-) Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C. (Can incubate tubes for 30 minutes, unless trying to grow DNA for ligation which is more sensitive. For ligation, leave tubes for 1 hour).
+) Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C.
-) Spread about 100 ul of the resulting culture on LB plates (with appropriate antibiotic added – usually Ampicillin or Kanamycin.) Grow overnight.
+) Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.
 ) Pick colonies about 12-16 hours later.