PCR Screening of transformants?

Koen De Smet k.desmet at nospam.ic.ac.uk
Fri Dec 5 08:01:06 EST 1997


Arnoud van Vliet wrote:
> 
> Dear Harry,
> 
> Colony PCR usually works fine in my hands using the following
> protocol:
> 
> 1. Aliquot 100 microliter aqua dest in 0.5 ml PCR tube
> 2. pick colony using toothpick; amount of bugs not very important as
> long as you're not putting in a whole plate -)
> 3. Heat 5-10 min at 95°C; I usually use a PCR machine as heating
> block. Shorter time probably fine; I can't be asked to try
> 4. Spin 5 min at full speed in microcentrifuge
> 5. Use 1 microliter as template  etc etc
> 

Here is my two pence worth:
1. Pick colony (or 10 ul liquid culture) into 100 ul water.
2. Add 100 ul chloroform
3. Vortex
4. Spin a few minutes
5. Use 1 ul in PCR
 
All my PCRs (also those with pure DNA) start off with a denaturing step 
of 5 minutes. Don't know how important that is.


Koen De Smet
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