trouble ampilfy genomic DNA - degradation, contamination or..? (Jun/02/2005 )
I have been trying to amplify a mouse' promoter for a while using BD2 advanced Taq polymerase.
Finally i got the right PCR product (1500bp).One month later I wanted a longer product (3kb) so I ran a new PCR and as well as a positive control the one that gave me just a band at 1500 (the reverse primer is the same..i just wanted to go further upstram!)
Surprise..almost the same pattern in both the PCR..with a faint band at 1500bp..and 4 lower bands.
i thought about contamination, so I changed water, tips, pipette, genomic DNA,Taq, buffer,fresh dilution of 10uM primer (from the same original stock)..nothing changed
there is no smear and it looks like amplification of something aspecific..even if before I blasted my primer against mouse genome
DNA is not too old (1 month in a fridge)..
I changed a lot of parameters in my PCR programme(annealing, elongation time,denaturation T) and nothing improved
So should I order new primers?
Last thing:I had as a water blank the whole master mix witout genomic DNA and i see just the primer at the bottom of a emty lane..is enough as a negative control?
thank in advance
is your polymerase optimised for long PCR, 3kb is quite a long fragment to amplify, I typically use expand long template from roche that is optimised for amplicons up to 15kb with great success.
I have no doubt that contamination is the problem. Please read this thread (the #20 post by "mantispid" in this thread) http://www.protocol-online.org/forums/inde...?showtopic=3895
sounds like your target sequence has a few sites that are similar in sequence to your primer target sites, hence non-specific banding.
I have some question for you, it might provide you some clue:
1. Did you BLAST your primer to chech their specificity?
2. How did you change your annealing temperature? Did you carry out a
gradient annealing temperature?
3. What is your MgCl2 concentration?
If your primer is unspecific, you should change it. Primer is mother of all PCR : )
If you carry out gradient annealing temperature, you should see the intencity of the unspecific band decreases.
If nothing change, use lower MgCl2 concertration (1.0mM minimum) using the highest annealling temperature that you got from your gradient.
Or else you can try to run at 0.8 - 0.6 X PCR buffer instate of 1 X.
The whole idea of doing all this is to decrease kation concentration in you solution which help to increase stringency in amplifying large fragment.
Finally, after doing all these still give you a problem, I would suggest you cut those unspecific band out, do a gel purification and send for sequencing. This will tell you exacally where went wrong.
Good luck to you.
Methylink, my plymerase is fine and can amplify up to 25kb
Hadrian, yes i blasted my primers and they gave my olny the seq i was looking for; MgCl2 conc is secret..is in the buffer they sell you, finally i just changed of 1 degree each time (up or down) up to 3 C in different reaction>>no gradient
PCRman..I attach a picture..still does it look contamination??
I'm getting really scared about the answer of bob1...
I try again one time...then i'll order different primer
thank you very much to everyone...plenty of good ideas
If you don't get anything in your negative control then I doubt you have a contamination problem.
Have you tried single primer controls? I've had cases where only one primer in the PCR mix gives you a nice band...
Thank for your gel photo, your gel tell alot of information. Is a good case study for me
First, that is not 4 extra bands beside the 1500bp, is more than that. I would say 6 bands. The consistantcy tell us that there is some extra oligonucleotide inside. Either your DNA or you primer had degraded.
This is because, when you have some DNA degraded inside you PCR reaction, the small peace of DNA can serve as primer and initiate a PCR reaction. Thus you get a lot of band instate of single specific band.
Judging from your gel,since your specific band is still consider dominant than others, I think is your DNA degraded. But you still have to do alitle work to varify
Could you please re-extract you DNA and run again? If with the new DNA extract, you get single band that mean your previous DNA stock had degraded. If the samething happen, might be your primer degraded. Then you have to re-aliquote your primer.
Can I know your DNA extraction method and what you use to elute your DNA?
Did you repeat thowing your sample?