Colony PCR - (Jul/12/2007 )
I am having problem in my colony pcr. i have tried more than a dozen times now over three months and have not gotten a single band. i have been uanable to figure out what is going wrong. If anybody have receipe for colony pcr? need sggestions on how to make this work.
Thanks in advance.
i also faced the same problem before when i did colony PCR. then, i add some PCR additives to PCR mixture... aftre that my problem solved and i got nice, thick and single band!!!
either using 10% of DMSO in your mixture, or add 10% of total reaction volume of the following mixture (10% formamide, 10% DMSO, 10% glyserol)...
When you do PCR on plasmid DNA itself, do you get a nice band? (is your PCR optimised?).
Do you take positive controls when performing colony PCR?
How are you doing it exactly? People tend to do colony PCR in a variety of ways, so please explain, I got mine to work without any additives.
I always do colony PCR and it always work fine without additives.
How do you do it?
Are you using your own primers or M13(or similar)??
For screening sometimes I prefer toothpick miniprep, it's easy, quick and cheep.
I think it is important to tell us how do you do your colony PCR. There are alot of threads discussing about this. Go and check some of them out.
Again similarly, I use autoclaved toothpick to pick a colony for PCR. PCR is a very sensitive application. So just a lil bit of colonies will do.
I usually use autoclaved pipette tips for transforming colonies. I dissolve my colonies in 50µl of ddH20. Heat it at 94ºC for 7 min and then centrifuge to get rid the cell debris. I use 2µl of the supernatant as template for pcr reaction. No pcr additives, using taq polymerase.
94ºC for 5min
94ºC for 30 sec
60 º C for 40 sec 35 cycles
72 º C for 1 min/kbp
72ºC for 10 min
run in 1% gel
I just dip very little bit of bacterial to the bottom of PCR tube, and add PCR mix for PCR, that's all
Tip, the amount of bacterial should be as little as possible...
usually I use 25cycles PCR (10ul reaction) for confirmation of insertion in cloning vector
well firstly , how long is your PCR product? In my hands, colony PCR using Taq can only realiabily amplify fragments under 1kb in size. Above that, some primers pair fail to amplify... and thus probably need optimisation.
secondly I have notice your annealing temperature is 60 Celsius. That seems rather high. Would it be possible to take a look at your primer sequence?