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Revision as of 14:17, 7 September 2012

Digestion Protocol For Plasmid Backbone Using EcoRI and PstI

-Reaction-

- 8 uL DNA linearized plasmid Backbone (25 ng/uL)

- 1uL PstI

- 1uL EcoRI

- 2.5 uL Buffer 10x must be a common buffer for EcoRI and PstI (e.g. buffer H in Roche system).

- X uL Water

TOTAL 25 uL

Mix by pipetting when both enzymes have been added. Avoid vortexing. Enzymes are kept in cooler or ice throughout all experiments.

-- The digestion mixture is kept for 3 hours at 37 ° C

-- The mixture is kept for 20 minutes at 80ºC

Transformation Protocol Using Heat Shock

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

2) Turn on water bath to 42°C.

3) 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.

4) Keep tubes on ice.

5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 30 min. to thaw competent cells.

6) Put tube(s) with DNA and E.coli into water bath at 42°C for 90 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)or SOC media. 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).

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

10) Pick colonies about 12-16 hours later.

Ligation Protocol

-Reaction-

- "X" uL plasmid DNA*

- "Y" uL insert*

- 1uL Ligase

- 1uL Ligase Buffer

TOTAL 10 uL

The ratio that has to exist between the number of molecules of plasmid DNA and insert is 1:3 (the volumes depends on the concentration of DNAp and the insert).