Colony PCR

Colony PCR protocol

95 deg – 2 min  (needed to break open bacteria OR for a hot start polymerase)
95 deg – 15 sec
58 deg – 30 sec
72 deg – 45 sec- 3 min (45sec for <1kb, 1 min for <1.5 kb, and 2 min <3kb)
72 deg – 7 min
4 deg- forever

50ul rxn recipe*
5 ul 10X Taq rxn buff ( w/ MgCl2)
2 ul primer 1 (10um)
2 ul primer 2 (10um)
5 ul DNTP ( 2mM each)
0.2 ul Titanium Taq polymerase (5u/ul)
35.8 ul H20

*Make a master Mix ( Make enough for two more reactions than needed. Example if you need 10 rxns make enough for 12).  Aliquot 50 ul of master mix into each tube.

Setting up for Colony PCR
1) For each colony label (1) 15ml culture tube and (1) PCR tube
2) Mix enough LB+antibiotic for each 15ml culture tube.  5ml LB+antibiotic / 15 ml culture tube
3) Aliquot 5ml of LB+ antibiotic to each 15ml culture tube
4)Make a PCR master mix and aliquot 50ul to each PCR tube

How to add a colony to a PCR reaction
1)Pick a single colony with sterile tip (try to single out lone colonies to avoid touching multiple colonies with the tip)
2) touch the pipet tip to the 50 ul PCR reaction tube and tap the bottom of the pcr tube 2x
3) drop the entire tip into 15ml culture tube with 5 ml of LB with the appropriate antibiotic
4) Add 2 drops of oil to each tube before applying the PCR caps
5) Run the PCR reactions for 35 cycles
6) Run out 5ul of each reaction and analyze the gel (include positive and negative controls)
7) Grow culture tubes corresponding to positive bands on gel

 

———————————————-
MINIPREP version
To use for screening minipreps, substitute 1 ul (or less) DNA from a
miniprep for picked colony

50ul rxn recipe with Titanium TAQ
1 ul DNA from miniprep (1 ul is plenty, could be diluted up 100x)
5 ul 10X Taq rxn buff ( w/ MgCl2)
2 ul primer 1 (10um)
2 ul primer 2 (10um)
5 ul DNTP ( 200uM each Cf)
0.2 ul Titanium Taq pol (5u/ul)
34.8 ul H20

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