help on PCR! - (Sep/18/2008 )
I am trying to amplify a 3.0 kb product. I am sure the protocol and program I used is correct since I did an trial before this experiments. However, I got strong smear on the gel, even the blank control in the leftmost lane. Can I say it is because I used the contaminated water or PCR tubes?? Thanks a lot and I need your help here!
Assuming your PCR tubes have DNA template, then yup, looks like the negative control got contaminated somewhere. Also note, your pipette barrel could also become contaminated if you are not careful.
However, this still does not explain the lack of a define 3kb PCR product. Is this PCR different in volume size to your trial?
Sometime the volume of the PCR reaction can affect the outcome of the reaction. The heating and cooling of a large volume is more difficult to control and cycle quickly compared to a smaller volume.
Could you repeat your trial PCR and see if you are still experiencing problems.
If the negative control is still contaminated, you will have to change all your PCR reagents. It is easier this way, than trying to find out which reagent is contaminated.
PCR contamination will occur, it is only a question of when. So in an event of contamination you should have the ability to throw away all your reagents and start with a fresh stock without too much hassle. Thus the importance of aliquoting all your reagents; dNTP, buffer, MgSO4 solution, etc. (and use a new box of tips)
If the 3kb PCR product remains missing, we will have look at this problem again (from the looks of the gel, it appears that too much template DNA is being used)
Try to use a highest Ta and/or a minor concentration of Mg.