%GC of my primers (59%) is higher than %GC of my template (32%). Do you think that is the cause of undetectable PCR product?
To amplify a low GC template you can try lowering the extension step from 72oC to 60oC for 2 minutes
Thank you Dr. Potaczek,
My product has a size around 1600bp. When I did the PCR and I didn't see any band, I tried several solutions:
1- check for the presence of the gene that I want to modified and I found the presence of that gene.
But with modified gene,...
2- do a concentration gradient of DNA template but I didn't see any product except oligo band
3- do a Tm gradient
4- Try a hot-start pcr
5- Increase the annealing time to 1 minute
6- Add Mg2+ to PCR reaction
I hope that you can understand my problem...Thank you so much
Use DMSO at ~5%, or other PCR enhancers, and or a polymerase for high GC organisms, think Kappa have one
To amplify a low GC template you can try lowering the extension step from 72oC to 60oC for 2 minutes
-To Dr. Potaczek: My purpose is to modify a gene which can be present in genome of bacteria...So "1" showed that there is the presence of the gene in my cultured bacteria.
-To Dr. O'Donnell: thank you for your advice, I will try it
-To Doyle: thank you, but my template has low %GC
Hoang
It may be possible that there is unspecific binding which results more than one band in agarose gel. You may also check the complementarity of primers. There is the possibility that primer can form primer dimmer.
All the best.....
I am agree with Dr. Amanda. I got the good amplification in one of my PCR product of about same length with low GC content. Also I am agree with Dr. Danial, try to change primer pairs, you might get results.
Good luck....
Hi,
The gene you are amplifying has large size, so use extension step to 60 to 80 sec
Also use new aliquot of primers or new set of primers. Try different conc. of primers, template, different annealing temperatures etc
use high fidility PCr kit or reagents.
Thanks
May be there is problem with annealing temp. So you can go for touch down PCR to address this. if your DNA was extracted long back then use BSA.
In my opinion, your target PCR product is too long. You will certainly increase the potential of fail amplification due to the length of the product. I suggest you to re-design two pairs of primers which amplify first part and second part of your target sequence, after that you can combine both of the part using a third pair of primer amplifying all region of your total 1800 bp. Hope this helps and good luck.
may be there problem with the primer complementary with the bacterial genome and annealing temperature.
Hi!
It seems that the problem in the extension time. One minute is too little for the length of your targete gene. Standard speed of the most polymerases is 1kb / 1 minute. And is doubtful idea about many hybridization places. Typically this leads to the formation of a wide field on gel, but not to complete absence of any bands.
It does not make sense to me that the primers have higher GC content than the sequence you are trying to amplify. Basically there is very low annealing capability of your primers.
It could be the annealing temperature. Try to optimize your annealing temperature. You can try 50, 55, 60 and 65 oC, and hopefully you will find the optimum annealing temperature. Also, run your DNA on agarose gel before you PCR, just to make sure that you have DNA extracted.
Hi，I suggest you design a new pair primer using vector NTI, you can check the primer is good or not using this sofeware
i think this is not the reason. try variation in annealing temperature.
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