PCR in 25 uL is it sufficient? - (Mar/17/2006 )

Hello,
I work on natural colony of cyanobacteria.
I have to realise 6 PCR with a single colony, so I havn't many DNA to work.
I use FTA paper for DNA extraction and it allows to perform my 6 PCR with a single colony.
Before I put 4 colony in FTA paper to be sure to have enough DNA to etablish these 6 PCR and results were great.
Now with a single colony I have results but not constantly.
->so I think it's due to a lack of DNA in PCR.
How can I increase it.
I was thinking using 25ul PCR instead of 50ul to increase DNA concentration!
Is it possible? Is it a good solution? Has somebody any others solutions?

Cheers

I routinely do PCR in 10 ul reaction. Some even use 5 ul. It all depends on your PCR machine. If it can hold 0.2 ml tubes, why not try less volume? I don't think template is a problem in your case. In theory, you can amplify your target from one positive bacteria.

If you think it's because of lack of DNA, why don't you try to optimise it a bit further so you can detect less copies of your target DNA? Lowering your volume will not be a problem (but I do that mostly to save money).

for colony PCR I use 25uL following EUROFAN program.
i have no problems

I actually don't think the volume for PCR matters as long as the concentrations are accurate.

I think the reason why people don't go below 10 ul is because you start running into more pipetting error...maybe not, though. I've never tried a PCR reaction below 10 ul :-p.

I usually do PCR with 25 ul.

-Matt

Hi
I usually do PCRs in 25 ul. Maybe just try to increase the amount of cycles or decrease the annealing temperature (but as you said the method you used worked before). you may add more DNA to the PCR to or prepare the templates for PCR in TE (then you make sure it does not get degraded as fast). Sometimes making smaller dillutions (like 10 x, 20x) of the quite "dirty" template after DNA isolation helps because many substances can stop PCR.
greetings