Smearing of the PCR Products - (Jun/07/2006 )
Hello,
I have been trying for the past few weeks to get a band from the PCR products. But all that i have got is a smear from the loading point till the point the gel has run. I changed my chemicals and my primers and also tried not using BSA. Still the results did not change.
Could anyone suggest me what should i do...... Its urgent....
Thank you in advance. 
You could be adding too much template to your reactions if you are amplifying from genomic. Dilute your template and try again. Use anywhere from 1ng - 10ng genomic / rxn. Load less on the gel as well.
Hi
i was also facing same problem from last month , keep one negetive control 2 find out contamination.
I have tried different concentrations of the DNA and have diluted the sample too but the results dont change. Intitially the positive control gave results but now even the positive control has a smear.
Currently i am doing a pcr with three different positive controls to see if they work...
i was also facing same problem from last month , keep one negetive control 2 find out contamination.
I have always kept a negative control. The negative control is negative n does not show a smear.
try decreasing the Taq in your reaction mix
You can also try a higher annealing temperature - what temp are you using now?
It also sounds like you have a contamination problem. If you need a quick fix - if you can, replace your taq, buffers, water, etc. and start over. If you see the problem again, its in your template or primers. Try to clean it up your template with a dna cleanup kit or phenol extraction, something like that. If its in your primers - you'll probably need to order a new batch.
I don't know if you have added Mg2+? if so, to decrease Mg2+ in your reaction mix might work.
It also sounds like you have a contamination problem. If you need a quick fix - if you can, replace your taq, buffers, water, etc. and start over. If you see the problem again, its in your template or primers. Try to clean it up your template with a dna cleanup kit or phenol extraction, something like that. If its in your primers - you'll probably need to order a new batch.
I am using the anneling temp of 62. I have also changed all the chemicals n tried the experiment. But all invane...
But i have now got a a band for the positive control...... so i guess i will repeat the DNA extraction again and repeat the PCR.
As I posted earlier, I think the problem is with the Taq. If you see the NEB techincal resources ( I don't remember exactly where-maybe with their vent pol) they mention to decrease the Taq quantity in the reaction mix, if we have such a kind of problem. or change the Taq completely.