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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