For screening colonies following ligation and determining DNA orientation (i.e. you can use a forward oligo from inside your gene and a reverse from the vector sequences downstream).
1. To each PCR tube add:
7. Prepare 0.7% agarose gel in 1xTAE containing 0.5 µg/ml ethidium bromide
8. Load 12µl of each sample on gel (prepare the sample as follows: 10 µl of PCR reaction + 2 µl of 6x loading buffer). Run gel at 100 mA.
· The time of expansion of the DNA fragment should be 1 min per 1Kb DNA
· Most enzymes won't produce fragments larger the 4kb.
· Any plain taq will do (we use “biotaq” obtained from Origolab -Yair Patish)
Reagents and solutions
50x TAE (per liter) 242 g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA pH 8.0
6x loading buffer: 0.25% bromophenol
0.25% xylene cyanol FF
40% (w/v) sucrose in water
Store at 4oC