mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 2
100% ethanol before being dried at 37°C for 10-15 minutes. The dry pellet was then
resuspended in 50μl SDW.
2.4.11- Ligation
The vector backbone and the DNA insert were usually mixed in a 1:3 ratio and treated with
1μl QuickStick DNA ligase (Bioline). The reaction included 5μl of 4X ligase buffer and was
made up to 20μl with SDW. The mixture is incubated at 4°C overnight in a water bath. 10μl
of the reaction is then transformed into competent E. coli cells.
2.4.12- Agarose gel electrophoresis
DNA (including plasmids, and digests) were run on 1% Agarose gel with SYBR-safe DNA
gel stain (Invitrogen) at 5µl per 100 ml agarose. To 20µl of DNA, 5µl of loading buffer
(0.25% bromophenol blue, 0.25% Xylene cyanol FF, 15% Ficoll type 400 [Pharmacia], made
up using 10ml sterile water) was added, and 10-15µl was finally loaded on the gel. Bioline
Hyperladder I was used as the marker for these gels. The gel was then run in a tank of 1X
TAE at 90V for 1 hour (for a 10 well gel) or at 110V for 1.5 hrs (for a 24 well gel).
2.4.13- PCR
Colony PCR and gene amplification
The PCR mix for screening and gene amplification is as follows:
- 10 NH 4 reaction buffer 5μl
- 50 mM MgCl 2 1.5μl
- dNTPs (100 mM stock) 0.5μl
- Primers (forward and reverse) 1-2μl
- Taq DNA polymerase (Fermentas) 0.5μl
81