Colony_PCR_screening

Colony PCR

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:

5 µl of 10X PCR buffer
1 µl of 10 mM dNTP mix
1.5 µl of 50 mM MgCl2
2.5 µl of primer mix (1.25 each 10 µM stock)
0.5 µl Taq Polymerase (2.5 U)
Add DDW to a final volume of 50 µl

2. Mix the contents of the tube by gently tapping the tube.
3. Pick a colony from the agar plate into the reaction mix with a sterile toothpick.
4. After diluting the colony in the PCR tube, drop the toothpick into a snap-cap tube with LB containing the appropriate antibiotics.
7. Perform PCR as follows:

93^oC for 3 min

94^oC for 45 sec
55^oC for 45 sec x 35 cycles
72^oc for 1 min

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.

Note
· 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 blue
                            0.25% xylene cyanol FF
                            40% (w/v) sucrose in water
                                               Store at 4^oC