Preparation of pUC 19 (Done with Hank)
- Took out ON culture of pUC19-containing cells (innoculated on 12 June 2009).
- Note that the culture rotor was not spinning - tube may not have been well-aerated.
- Mixed the entire 80µL of RNaseA into Charge-Switch Pro Resuspension buffer.
- Aliquoted 1.25 mL of ON culture each per microfuge tube (4 in total), spun down at max speed for 10 min.
- Followed ChargeSwitch Pro miniprep protocol from here on; combined the harvested cells into two tubes (one from Hank, one from me).
- Nanodropped the samples:
| Tube
|
[DNA] (ng/µL)
|
A260/280
|
| pUC19 (1) |
31.11 |
1.26
|
| pUC19 (2) |
26.36 |
1.27
|
- Stored at AMBLE -20ºC freezer
- Moved to Lagally Lab -20ºC freezer, labelled:
- pUC19 HYEM(1)
- pUC19 HYEM(2)
- Made glycerol stocks of these cells (250µL of 60% sterile glycerol + 750µL of ON culture)
Results of Mutagenesis
- Hank noticed that the media was cracked; could be too thin.
- Took plates out from AMBL 37ºC incubator @ 2:45 PM.
- Observations
| Plate
|
Colony count
|
Colors
|
| Positive (conc.) |
0 |
n/a
|
| Positive (dil.) |
0 |
n/a
|
| Negative (conc.) |
TNTC |
White (all)
|
| Negative (dil.) |
135 |
White (all)
|
| Control (dil.) |
20+1 |
Blue+White
|
| Control (conc.) |
322+33 |
Blue+White
|
- Plates were parafilmed and stored at AMBL4ºC.
|
iGEM Project name 1
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