Removed Transformation Plates from 37C - Colony Count
Plate Plate Marker Count Notes insert only Cm 0 insert only Kan 3640 very small cPCR #6 Cm 240 very small cPCR #6 Kan 532 normal sized cPCR #6 (-) control vector only Cm 26 very small cPCR #6 (-) control vector only Kan 0 cPCR #8 Cm 1600 very small cPCR #8 Kan 2400 normal size near center, very small on peripheral cPCR #8 (-) control vector only Cm 11 very small cPCR #8 (-) control vector only Kan 5 very small
LC'ed 6 colonies each from cPCR #6 & #8 S.A. ligation of F+B+CPX+B0014 into pSB1AC3 in B-R cells
- 37C @ 10pm
Transformed Ligation of F+B+CPX
- Used Joey's B-R e.coli cells (electrocompetent)
Electrocompetent procedure: 1. After ligation/denaturation, use pore to filter DNA (10µl) in water in petri dish 2. Add DNA to 50µl of cells 3. Add mixture to cuvette 4. On machine, set to EC20(for e.coli) and measurement to time in ms 5. Load cuvette with knob on right, ensuring that all fluid is on bottom 6. Insert cuvette so that both metal electrodes are touching metal sides of cuvette 7. Press Pulse button 8. Time measurement should be approximately 6 ms (ours were around 5-5.3) 9. Immediately add 1mL of LB to cuvette and transfer mixture back into eppendorf with LB 10. For growing cells on Chloremphenicol media, let cells recuperate for 1 hour. This step is not necessary for Ampicillin.
- Plate all 5 samples (cPCR6, cPCR8, -cPCR6, -cPCR8, F+B+CPX insert only) on both Chloremphenicol and Kanamyacin plates for antibiotic screening. Hopefully colonies will grow only on Chloremphenicol plates.
- Plates placed in 37C at around 12am