E coli ChemComp prep

Mix & Go E. Coli transformation kit

Adapted from Mix & Go mini instructions from zymo. Possibly works with SOB as well. Do not use at home as it absolutely requires refigerated centrifuge. This procedure is for 50ml of E coli culture and makes approx. 50 comp cell reactions at 50ul.

DAY BEFORE
1. Prechill tubes
-Prechill 2 50ml (-20c)
-Prechill 48 1.5ml (-80c)
2. Put in overnight culture of E coli
-1ml of E coli, doesn’t matter media

DAY OF
1. Chill centrifuge
2. Add 0.5ml of saturated E coli cuture to 50ml of ZymoBroth and incubate at 30c for ~4 hours
-Growth at 30c increases competency of cells
-Take cells out between OD 0.4 and 0.6
3. Transfer 50ml flask to ice for ~5-10min
4. Pellet cells at 2300g for 10min at 4c
-Will have to split between the 2 prechilled 50ml tubes
5. Resuspend cells in 5ml of 1x Wash Buffer
-2.5ml to each chilled tube
6. Pellet cells at 2300g for 10min at 4c
7. Completely remove supernatant and gently resuspend cells in 5ml 1x Competent Buffer
-2.5ml to each chilled tube
-Important to completely remove supernatant
8. Pour into reservoir at use multichannel to resuspend 50ul into prechilled tubes
9. Quickly freeze

NOTES
-Usually get 10^5-10^6