Weird PCR results from 2 different samples - (Dec/13/2006 )
I have two different samples. Sample 1 is my genomic DNA. Sample 2 is my cloned plasmid. Using similar PCR conditions and solutions, it seems that they have two distinct different PCR product even though similar bands were observed.
The only key difference is the amount of DNA and types of template. For genomic DNA, about 140ng of DNA inside 25ul of solution. For cloned plasmid, I did the direct PCR method for colony screening.
The final results are from Sample 1, faint desired bands with a lot of primer dimers present. Sometimes at low temperature it might appeared to be unspecific binding. For sample 2, clear and strong desired bands were observed with less primer dimers.
So the thing that is puzzling me is why is there such a difference? Is it because it is harder for primer to bind in the genomic DNA compared to cloned plasmid? What can I do in order to optimize my PCR amplification from genomic DNA? I did gradient, conc of MgCl2 and amount of DNA. It is still the same.
Thanks for reading and helping. =) Really appreciate it. Thanks guys.
There is most likely a huge difference in template copy number between the two samples that could account for this. Also, make sure you are not comparing a cDNA to a genomic DNA. You may have differences in intronic sequences.
It sounds to me that there is not enough time for the gDNA strand to open so the polimerase could bind and amplify. Add more time at 95C. Remember that the gDNA is much bigger than the plasmid so the plasmid has plenty of time for amplify and the gDNA not.
Thanks for your reply. and yea, I am comparing plasmid DNA and genomic DNA. So wouldnt be a problem when it comes with intronic sequences.
hmm... it sounds very logical. Thanks. However, for initial denaturation, I had already set 15 minutes at 95C. Do I need to increase more? What is the limit for it? Thanks once again.
and additional factor is DNA quality.
if is often the case that genomic DNA is contaminated with low levels of "inhibitors of some nature" which reduce PCR activity. Plasmid DNA from e coli, don't suffer this problem.
15min of denaturation is really long. I would normally denature no more then 5 mins. It is quite possible that your DNA is being trashed quite badly by the long denaturation step.
The initial denature should be around 5-8min, 15min is toooooo muuucchhhh!!!!!! check the temperature if you are using a hot start enzyme the temp should be 95C or more or not enough enzyme will be free in the mix. When you are in the cycles per se denature for about 30-45seconds, anneling for 30 sec-1min (depends size) and extend 45sec-1min. #cycles 25-35 again depends size of the product and quantity of tamplate. About quality of sample check that your sample is free of ETOH, sometimes if you don't dry well the sample after precipitation the ETOH will inhibit the reaction. I prefer to dry the samples under vacumm. Another posibility is that when you pipet the sample you are not taking the quantity that you expect always vortex the sample. Hope that this help if not email me and send the photo.
is 15 minutes is way too much? I know it is long. But i think it is ok considering if i am doing colony pcr when i have to break the cell wall.
I will try again next week. It is weekend now. Anyway, thanks alot for your opinions. Really appreciate it.
Thanks merlav and perneseblue
check this maybe could help http://www.upstate.edu/biochem/amberg/prot...colony_pcr.html
colony PCR only needs 5mins. There is the heat and the detergent in the polymerase buffer. Both act toget to break the cell wall.