Screening Bacterial Colonies

 

 

Comments:

 

 

 

 

Procedure:

 

  1. Heat up plates with colonies to be picked at 37 C.
  2. Prepare re-plate by drawing grid (with numbers) on new Amp+ plate. Warm up at 37 C.
  3. Put 5 mcL of Millipore water (fresh) into each PCR tube.
  4. Pick each colony, dip into appropriate tube, and then spot on new re-plate. (new colonies will show up between 6-8hrs.)
  5. Prepare master mix accordingly: (multiply by #samples +1)

 

1 x pre-mix

_

25 x pre-mix

11.5ml
2
ml
0.6
ml
0.5
ml
0
.1ml
0.1
ml
5
ml
0.2
ml

H2O
10 x PCR buffer
MgCl2 (50mM)
10mM dNTPs (i.e. a mixture of all four at 10mM each)
primer 1 (100
mM)
primer 2 (100
mM)
Template
cheap Taq

287.5ml
50
ml
15
ml
12.5
ml
2.5
ml
2.5
ml
- 
ml
5
ml

 

 

 

 

 

 

 

 

 

 

 

Note:  Role of MgCl2

      Magnesium chloride is the preferred method of adding magnesium to a PCR experiment. Thermostable polymerase requires the presence of magnesium to act as a cofactor during the reaction process. Its role is similar to that of a catalyst in that the magnesium is not actually consumed in the reaction but the reaction cannot proceed without the presence of the magnesium. Too much magnesium causes rapid reaction and error prone polymerase. Too little causes slow reaction.

 

 

  1. Add 15mcL of master mix to each PCR tube (already with bacteria added)
  2. Set up the following PCR reaction in the machine. (Check for pre-set reactions in machine)

 

Cycles

Temperature

Duration

 

 

 

1

94C

60 seconds

 

 

 

25

94C

30 seconds

55C

30 seconds

72C

1 minute

 

Note: This will take less than 2 hrs. to run. During this time, make an agarose gel (probably between 1.5-2%) to run out the samples.