Team:Stockholm/12 July 2010

From 2010.igem.org


Contents


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!)

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.





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/