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Team:Stockholm/12 July 2010
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Andreas
Clonings
Gel extractions and DNA clean-up
Continued from 9/7
Gel extracted DNA samples (pEX, BBa_J1893x, SOD and yCCS) and digested DNA samples (pSB1x3) stored in -20°C were purified using the E.Z.N.A. Gel Extraction kit.
Nanodrop measurements revealed extremely low or even nonexistent concentrations of DNA in purified samples. I decided to go ahead with the ligations anyway, as I was confident that there was indeed (correct) DNA present in the samples.
Ligations
- Ligation strategy:
Vector Insert pSB1A3 BBa_J18930 pSB1A3 BBa_J18931 pSB1A3 BBa_J18932 pSB1A3 SOD pSB1C3 BBa_J18930 pSB1C3 BBa_J18931 pSB1C3 BBa_J18932 pSB1C3 SOD pSB1C3 yCCS pSB1K3 BBa_J18930 pSB1K3 BBa_J18931 pSB1K3 BBa_J18932 pSB1K3 SOD pEX BBa_J18930 pEX BBa_J18931 pEX BBa_J18932 pEX SOD pEX yCCS
- Procedures
- All insert samples, as well as the pEX vector sample, were concentrated in a vacuum evaporator prior to ligation
- 2 μl vector
- 5 μl insert
- 10 μl 2X Quick Ligase buffer
- 2 μl dH2O
- 1 μl Quick Ligase
- Incubation 10 min in "RT" (30°C due to the extreme summer heat these days!)
- All insert samples, as well as the pEX vector sample, were concentrated in a vacuum evaporator prior to ligation
Transformations
Ligated constructs transformed into Top10 and plated onto LB agar plates with relevant antibiotics. pEX.BBa_J1893x constructs also transformed into BL21 cells and plated onto 100 μg/ml Amp with 1 % glucose. Plates incubated ON in 37°C.
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