Have two sets of primers to amplify two separate genes from the same genomic DNA template. I have been using fresh reagents. while one primer set is showing non-specific amplification (including my amplicon of concern, but less intense), the other is not showing any amplification at all. However, I do observe utlilization of the primers. Also, when I tried gradient PCR using low volumes (10ul), I did observe minor amplification in case of the other set but scaling it up led to no amplification.
hi
did you test in silico PCR to see if primers are specific?
if no please try the online version of the UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=575437527_e5IGp6x0ly23Dd8wgEL9gfLAMaWN). change genome and assembly if you're not on human.
fred
Hello Frederic, thank you for your reply, actually I did test the integrity of my primers using online tools and Tm wise and hairpin structure wise they look alright. However, the link that you have suggested does not have the genome of the bacteria that I am working with so could'nt try that. Also I have tested both Taq and Phusion polymerse and both behaved similar. I have a feeling I should may be play around with the annealing and extension time. What do you suggest?
OK, don't know the bacteria but DNA still DNA.
what I would do is a touch down PCR with 20 cycles 0.5°c/cycle beginning 10°c over your Tm and additionnal 30 cycles at your Tm. time is not often the limiting point (think at the skin of your face that is regenerated every days (means 3MpB in 24hrs by the DNA pol...)). test 3"/5"/15" for step durations. check if it's necessary to activate the Taq (that implies a 12' step at 95°c), quick answer you'll get.
fred
agreed with Frederic Lepretre. If you still fail to amplify your target then you need to redesign your primer
To make band intense i do increase the concentration of MgCl2.But that wont solve the problem of nonspecific amplification.
If you can kindly share the gradient gel pic, i can comment more.
attach here or msg me
To get both products amplified equally they need to have close to the same Tm under your PCR conditions, so size must be similar given the same GC content. Although you indicate you've tested this, different tools can give different results so try a few and see how they compare. Are you getting primer dimer product from the underperforming primer set? If so you'll need to optimise your conditions, particularly use >95o denaturation if you aren't already and use a high Mg concentration and a long extension time (use 1 min/kb) to try and force some product that you can then optimize for yield and specificity. Sometimes primer sets interact with each other in unexplained ways (I have developed a lot of 5-10 plex reactions) and you can waste a lot of time and reagents trying to optimize, so if after a few attempts you have no luck as others have said, redesign. Are they labelled at all?
Hello All, Thank you very much for your suggestions. I appreciate it. As Frederic and Abdul had advised I did try the touchdown PCR and unfortunately it did not work, in fact in case of the non-specific amplification set, I am getting a more amplified non-specific band instead of the band of my interest. and in case of the underperforming primer set, I still see no amplification, I had set up two separate reactions of 10 and 50 uls each just to check if the machine is not working efficiently. I am thinking of going ahead with redesigning my primers.
David as suggested by you, I have reanalysed my primers with other freely available softwares and they do seem to be fine, however, the Tm for the two primers in a set are varied by 7 in first and 10 in the second set, as far as GC content is concerned, they are all around 40%. my only apprehension is I need to increase the length of the primer in this case which I hope will not cause any problem....will keep you guys updated....
hi
when I design primers I apply some unescapable rules, as no more than 2°c Tm difference between primers, amplification Tm between 55 and 60, no hairpin and less than possible dimers, and almost one G or one C at 3' and 5' ends. each time I after run in silico PCR in UCSC to ensure that the amplification give only one target, and check for SNP in the primer sequences to avoid low amplification in some samples.
fred
I suspect the difference in Tm is an issue. GC is Ok but as Frederic says, no more than 2o difference in primers. Both sets need to have close to the same Tm to operate in the same reaction.
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