PCR Optimization Question - Non-specific smearing around target level? (Oct/17/2008 )

Hi everyone,

I'm working on a project currently and in a phase where I'm trying to amplify a specific product through standard PCR means. I continue to not get optimal amplification but get faint smears around where my target gene would be for all samples (minus the negative). This persists through various treatment (lowering annealing temp to 48*c, increasing annealing time by a few seconds, increasing annealing temp up to 53*c, using formamide up to 3%, etc., using BSA, hot primer use, etc.).

I was hoping that someone here might have a clue what this might be/how to turn those faint smears into nice tight bands. There's a link to a picture of the gel for one of the reactions with the offending product at the bottom of the post. Any help would be greatly appreciated, I'm currently at a loss as to how to treat this problem.

i495.photobucket.com/albums/rr313/element522/101608-highanneal_formamide.jpg

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Hi Dan,

Okay, so I'm assuming that the blazing band in lane 10 of the bottom gel is a positive control? :-)
The other lanes look like there's almost nothing there, to tell you the truth. What is the Tm of your primers?? 48°C is extremely low -- 53° is still pretty low. Are these RT-PCR products? How many cycles are you running? Is the positive control template also cDNA? A couple of your lanes look degraded (4 and 7 on the bottom).

To tighten "smeary" bands, it's usually a matter of increasing the gel percentage and adjusting voltage -- but judging from the sharpness of your ladder and your positive control, the gel is not the problem. It looks to me like a lack of DNA is the problem.

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Could try varying the Mg2+ conc.
Tell me your PCR cycle

You could try using mastermixes they have some enhancers which could help u out in the mixes to optimize PCR.
for me, master mix green 2x from lucigen got a good record so far.

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if i got this right: lane 13 in the 1st gel / lane 12 second gel are markers, and all the rest lanes except lane 10 on the second gel are your reactions.
how much sample did you load from the reaction to each well?
what is lane 10? is it a different template just happens to be right size or something?

anyways, I think if your pcr rxn ends with almost no product (almost looked like the loading buffer dye to me), it's probably either the template or primer, check to see if you actually have dna in the reaction, just load a gel it's like 15 min easy check. if you put in more than 0.2ug in the pcr reaction then it's most likely you have wrong primers in the reaction, check the tm value, try to make it to between 60-65C and 5'/3' are within 1C of each other. also make sure you added everything right and enzyme is active.

try the following: add 0.6-0.8ug of dna, 1ul 10um 5', 1ul 10um 3' primers, 2ul 10mm dntp, 5ul 10x rxn buffer, water to make it 49ul total and add 1ul pfu polymerase, double check your tm, run the annealing @ tm you calculated, extension at 72 for 2min, just to see if you can get a blazing product first. then you can optimize it w/ formamid, q-solution or whatever.

if you can't get tm high enough, add some restriction sites will work.

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BTW , adding restriction sites will NOT raise the true tm of annealing primer.You might see it on the tube that the manufacturer sent to you because they assume that all the bp will anneal to the template but clearly this is not the case. all in all, it raises it artificially .. in the end u are back to redesigning a better set of primer using Primer 3.

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Hi thanks for the replies everyone,

My primer Tm's are at 51 & 52*c, which is why the annealing temp is ran so low. Lane 10 is the positive control, the rest are all unique samples. And my cycle consists of an initial hold at 94*c, denaturation at 94*c (30s), annealing at 50*c (30s) & Extension at 72*c (90s) (30 cycles), followed by a hold at 72*c for 10 mins. The annealing temp of 50*, is known to work well for what I'm amplifying (bacterial 16s), so I'm hesitant to raise it/lower it too much, especially since my primers are already well suited for that temperature.

The confounding thing is that I've had some of these reactions work before (in very tight annealing temp windows) and produce nice tight bands, and as you can see on the picture, there aren't any aside from the positive control. There's also definitely DNA in the samples (lanes 4 & 7 on the bottom are most likely a result of too much DNA I believe). And the DNA seems to be of at least fair quality after running it through a gel. So knowing the DNA is there, I've got my dilutions pretty much targeted (based on other working reactions from similar samples), yet for some reason I can't seem to achieve optimal reactions with these that are on the gel. Is it possible that non-specificity could be an issue b/c of the lower annealing temp?

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if you have lots of non-specificity binding, you'll still get some obvious bands, the problem is that you have no band. If your primer tm is 51-52 deg, you should do annealing at 52 deg, and you can increase the extension to 2 min. for 35 cycles. and also you only stick to the same procedure when it works, not when it doesn't work. In this case, I think the best thing you can do is to change stuff dramatically to get a product.
You need to determine the concentration of your template as well which can be done using a spectrometer or low mass ladder. And you need at least 0.4ug in the pcr if you want a really bright product. These are realy simple stuff that'll take max 2 days (including midiprep).

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