problem in Pcr amplification - (Jan/27/2009 )

Hi all,
Iam rigourously facing this problem since a couple of months. When I performed the pcr, I got te correct amplicon. To confirm it I repeated and got correct for the second time ,and the third time it was correct but with one non-specific band.sometimes I get correct and sometimes correct but with the non specific bands.what could be the problem.

appreciate your suggestion

Hello

Justy increase the annealing temperature by 1 or 2 degrees to get rid of the non specific band.

Best
TC

novagen on Jan 27 2009, 06:29 AM said:

I think there is contamination in your 3rd batch, given those non-specific bands were not present in the 1st and 2nd runs and given you were using the same protocol. Run a negative control to test for contamination or ignore the non-specific band and cut out the amplicon band from the gel to continue cloning.

HAve you checked the reagents to make sure they are fine. If you have the right band or not you need to sequence. Running it on gel will only give you an iea of the size of the amplified product.

You could reduce teh amount of DNA template used to reduce the nonspecific bands. I have experienced this scenario before. Try and see what happen

Make 1:10 dilution or perhaps 1:5

BTW, service to women is service to God too And only then your PCRs will work reliably!!

As previous posters suggested, run a neg control (neg controls should always be run though), and try diluting the template, as too much starting DNA can either inhibit the reaction or drive non-specific amplification. Another alternative is that there is some sort of template in the sample which non-specifically reacts with your PCR assay, but in concentrations low enough to be at the threshold of sensitivity of your assay. This will then manifest itself as pos/neg results over a series of replicate runs. Good noews is that a good serial dilution should get rid of that as well, assuming your target template is of high enough load.

novagen on Jan 28 2009, 12:29 AM said:

hi all,
I want to to express plant gene in the yeast.
which is the best yeast cloning vector that can be used.
thanx a lot