Parashar Lab

Notes

 

This is the fastest way to screen bacterial colonies. Our PCR machine takes 24 tubes so I routinely screen 22 colonies + 1 negative + 1 positive control.

 

Choosing the primers

 

Ideally you want a primer pair that can only work if the correct construct is present eg. a vector flanking primer and a gene specific primer. However, this may not allow you a positive control (essential) so you might have to use both vector flanking primers instead. If you have enough space in your PCR machine you could do both.

 

Typical Protocol

 

Set up 24 PCR tubes each containing 5 µl H2O.

 

Touch a fresh toothpick (or yellow tip) onto a colony, dip it into a PCR tube, then streak it onto a fresh replicate agar plate using a numbered template (that is, all 24 colonies on a single agar plate).

 

I don't label the PCR tubes. I can tell what number they are and also whether they have been 'dipped' by their position in the rack/block.

 

Repeat this for the 22 (or whatever) colonies

 

for tube 23 (negative control) use a colony that is negative (or use nothing).
for tube 24 (positive control) use a colony that will yield a product with your primers.
If you don't have a +ve colony then use a tiny amount of plasmid DNA

 

Incubate the replicate agar plate at 37°C.

 

These streaked colonies will be visible within 6–8 hours so you can set up overnight miniprep cultures on the same day.

 

Set up a 25 x PCR pre-mix as follows:

 

 

 

1 x pre-mixComponent25 x pre-mix

11.5 µl
2 µl
0.6 µl
0.5 µl
0.1 µl
0.1 µl
5 µl
0.2 µlH2O
10 x PCR buffer
MgCl2 (50 mM)
10mM dNTPs (i.e. a mixture of all four at 10 mM each)
primer 1 (100 µM)
primer 2 (100 µM)
Template
cheap Taq287.5 µl
50 µl
15 µl
12.5 µl
2.5 µl
2.5 µl
0 µl
5 µl

 

Add 15 µl of pre-mix to each PCR tube.

 

 

 

PCR program

 

 

 

CyclesTemperatureDuration

194°C60 seconds

2594°C30 seconds

55°C30 seconds

72°C1 minute

 

This PCR should take less than 2 hours. While the PCR is running prepare the agarose gel ready to analyze the PCR products.

 

 

 

When you have the result you can go to the replica agar plate on the same day and set up miniprep cultures of the likely candidate colonies.