Transformation and Plating

Transformation Protocol
Transformations are used to insert the designed DNA into bacteria, which is then grown to multiply the plasmid DNA.

Heat transformation Protocol

1.	Make sure that the incubator (30/37C) and water bath (42C) are ON

2.	Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map. 3.	Take the DNA out of –20 frig, let it thaw

4.	Thaw the competent cells on ice for 7-8 min. 5.	Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. For Ligation add 1-5 ul

6.	Incubate the cells on ice for 30 min

7.	Heat shock the cells for EXACTLY 30 sec at 42 C water bath.

8.	Place on ice for 2 min.

9.     Add 0.9ml of 37˚ C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)

10.	Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min

11.	Spin down the cells at 6000 rpm for 1 minute (should see white clumps at bottom).

12.	Take out 0.85 ml of S.O.C. (so there’s only 50 ul of SOC left inside)

13.	Resuspend the cells.

14.	Plate all 50 ul.

15.	Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C. Can leave the cells in the incubator for up to 18 hours but no more