June, 28th
First time in wet lab
11 LB agar plates, 35 LB agar + Amp plates and 15 LB agar + Cm 12.5 were prepared.
*BBa_C0060 was resuspended from iGEM 2011 kit distribution, Plate 1, well 4K in 15 ul of ddH2O
*BBa_C0061 was resuspended from iGEM 2011 kit distribution, Plate 1, well 4M in 15 ul of ddH2O
*BBa_J04450 was resuspended from iGEM 2011 kit distribution, Plate 1, well 5E in 15 ul of ddH2O
*BBa_K081022 was resuspended from iGEM 2011 kit distribution, Plate 2, well 12N in 15 ul of ddH2O
For each of them 1 ul was transformed in 100 ul of home-made TOP10 competent cells.
Transformants were plated on LB agar plates with the right antibiotic and incubated over night at 37°C.
| colony chosen |
ligation name |
| I0-2 | I0 |
| I1-2 | I1 |
| I2-1 | I2 |
| I3-1 | I3 |
| I4-2 | I4 |
| I5-1 | I5 |
| I6-2 | I6 |
Other colonies resulted positives at the screening but were NOT sequenced and are stored in the iGEM 2010 cemetery box. These clones are: I1-1, I2-2, I4-1 and I6-3.
June, 29th
Inoculum of I6, BBa_J23118, BBa_J23110, BBa_J23114, BBa_J23116 from glycerol stock in 5ml LB+Amp. Cultures were grown ON at 37°C 220rpm.
June, 30th
PhaP-1 and PhaP-2 plates showed colonies!!
A colony from each plate was picked and used to infect 1ml LB+Kan. Liquid cultures of PhaP-1 and PhaP-2 were grown for 6 hours at 37°C 220 rpm and then used to prepare glycerol stocks. Remaining liquid cultures were re-filled with 5 ml LB+Kan an grown ON at 37°C 220 rpm for tomorrow MiniPrep.
Glycerol stocks of PhaP-1 and PhaP-2 are stored at -80°C in the iGEM 2010 Registry box.
Ligations were heated at 65°C for 5 minutes to inactivate ligase ad then tranformed in E. coli TOP10 home made competent cells. Plates were incubated ON at 37°C (we decided to incubate plates 5 hours longer than usual, because cell growth was already slower for I6).
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