Transformation

Steps

1. Make sure that the incubator (30/37C) and heat block (42C) are ON
2. Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI.
3. Take the DNA out of --20 frig, let it thaw
4. Make sure that all of the required reagents/DNA etc are present at the site of transformation before you take the cells out of the -80.
5. Thaw the competent cells on ice for 7-8 min.
6. Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. If transforming multiple plasmids, add 10 ng of each.
7. Incubate the cells on ice for 30 min
8. Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
9. Place on ice for 2 min.
10. Add 0.5ml of room temperature 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.)
11. Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min (shaking incubator)
12. Plate 100uL on one side of a plate
13. Spin remaining cells at 5000rpm for 1 min, pour off supernatant, re-suspend cells in remaining liquid (DO NOT vortex) and plate on other side of plate.
14. Incubate plates 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.