Transformation

WASN'T SOMEBODY GOING TO UPDATE THIS?  -bt 06/29

Steps

1. Make sure that the incubator (30/37C) and heat block (42C) are ON.
   1. Put water in the wells of the 42°C heat block.
2. Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI Make sure you're using the right antibiotic plates for your plasmid's resistance!
   1. Warm plates to 37°C.  Cold plates reduce transformation efficiency by an order of magnitude.
   2. Also warm 500 µl SOC per transformation.
3. Take the DNA out of --20 frigfreezer, let it thaw.
   1. Vortex DNA to mix, then spin down.  Make sure it is completely thawed out!
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 73-8 4 min.
   1. You want to add your DNA right as the last bit of cells' ice melts.  Even if it's still a little slushy, that's okay.
6. Add 1-2 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 eachinto the comp cells.  Stir with a pipette tip a few times, then put right back on ice.
   1. If you're transforming the result of a reaction (GG, LR, etc) add 1-2 µl of the reaction.  Don't add more: many of these reactions have additives that screws up transformation.
   2. If you're transforming plasmid DNA (from a miniprep), either (a) dilute it out so you add only ~10 ng of DNA, or (b) plate only 10 µl of the outgrowth – else you'll get a lawn!  Super-coiled DNA transforms super-efficiently.
7. Incubate the cells on ice for 30-40 min.
8. Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
9. Place back on ice for 2 min.
10. Add 0.5ml of room temperature S.O.C SOC (37° to RT) 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. .
15. Plate 100 µl for a reaction product, or 10 µl in a 100 µl puddle of water for a supercoiled plasmid.
16. Incubate plates upside down overnight 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.