24 replies to this topic

[rudzielec]

I've got to amplify some exons. Everything was ok so far - one single band on a gel,no probs with PCR. Because I've got to sequence all my PCR products (looking for mutation) I had to add M13 tag to my primers  - that'll be easier for people doing sequencing (just M13 primer will be needed instead of hundred different for each exon). Problem is that I'm getting one extra band (quite faint but it still is) in all exons when amplifying with that new primers. I just realised that extra band is always double size when you compare to the correct PCR product size...I've tried to increase Tm but finally I've got 70°C and still that extra band....I've tried adding DMSO, betaine etc. and no change. I do not want really do gel purification as I've got lots of samples and purification on column would save so much time. I've been told that my new primers should not change my PCR conditions.....and I should still get just one nice band on a gel....
Have you got any suggestion what else I can try to get rid of that double size band?
Thanks,

[HomeBrew]

Hi rudzielec -- welcome to the BioForums!

How big is your PCR product?  Can you adjust your extension time so only the shorter (correct) band appears?  Can you use another sequencing primer tag, other than M13?

[swanny]

I'd suggest you just try and get some sequencing done, and see how bad the noise actually is from this second band. If it is not too bad, the software should be able to discriminate.

The fact that it's always there, no matter what you change, suggests it might be something unrelated to the amplification reaction. How big is the expected product? The second band (apart from just 2x the expected band)? What template are you using? How was it prepared? Have you changed all of the reagents? Have you cleaned your pipettes / used someone else's pipettes / used someone else's machine? Have you changed PCR preparation rooms?

Most of all, could you upload an image of the gel?

[rudzielec]

Thanks for reply!
PCR products are 200-600bp. When I tried to cut the extension time down previously I was getting my correct band fainter and other unspecific bindings didn't disappeared completely. But haven't tried that for PCR with the M13 tag. The problem is that I can not use other sequencing primer tag I use gDNA as a template. I don't think that the problem is because of pipettes, reagents or machine because other PCRs with normal (without M13 tag) primers work very good. I've uploaded one of my gel images. That double size bands are quite faint but previously when I purified (on a column) PCR products with extra bands as faint as those, I had quite noise sequences in some of them. I don't have a proper software so sometimes it's quite hard to say if it's mutation or not I've got lots of samples so I can't spend lots of time contemplating if that's just noise or maybe mutation or repeating PCR and sequencing again If it'd be possible I'd like to have nice sequencing results

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[HomeBrew]

rudzielec, on Apr 9 2010, 04:40 AM, said:

The problem is that I can not use other sequencing primer tag I use gDNA as a template.

I don't understand -- is your M13 tag a 5' extension added to your primer?  Can't you just re-synthesize your primer with a different 5' extension?

[rudzielec]

People who'll be sequencing my samples want me to have M13 tag as they use it and it's easier for them-they don't have to change program or something I don't know....

[Mighty Mouse]

rudzielec, on Apr 9 2010, 06:51 AM, said:

People who'll be sequencing my samples want me to have M13 tag as they use it and it's easier for them-they don't have to change program or something I don't know....

You could always try http://www.genewiz.com/

I haven't used them yet, but I've heard it's easy. I suspect there are a lot of companies out there that will sequence your PCR product for you on the cheap.

MM

[rudzielec]

Problem is that I can't use other company to do sequencing....

[HomeBrew]

Okay, given all the restraints you have, the next thing I would do is find out what that larger band is -- slice it out of a gel, and have the PCR product sequenced.  Comparing the sequence of this band with that of the correct band should tell you why it's being amplified, and allow you to alter the homologous part of your M13-tagged primer to avoid it being amplified.

It may be that your M13-tagged primer has to be purified by your primer synthesis company using HPLC or gel purification to remove truncated synthesis products, insuring that you only have full-length primers.

You may also have to fool around with your PCR mix (e.g. Mg++ concentration) or your parameters (e.g. number of cycles) to stop amplification of your spurious product or minimize it to a degree that it doesn't interfere with sequencing.

[rudzielec]

Probably I'll have to do gel purification for all my PCR products as I've tried to do what I could with PCR and I couldn't get rid of that extra band. I'm just wondering if there is any chance that the larger band will not messed up sequencing. Maybe I need better software to analyze the sequencing results but it needs to be free

[Trof]

If the longer band has same primer sequence as your product it could interfere with the sequencing, it will increase background and it will be more difficult to find a mutation if it's only in a portion of cells, or it would render the sequence unreadable if it's more concentrated. You should try to get only single band to sequence.

A have another idea, run a first round of PCR with your untagged primers, you should get a clear single band as you said, then dilute it many times (like 1:10 or 1:100) and amplify with the M13 tagged primers. There should be no non-specifities in that second round. Only drawback may be, that more cycles means higher probability to get an polymerase error, but you can try  lowering the number of cycles in the first PCR.

[rudzielec]

I'm going to try that today and we'll see if that helped Thanks!

[rudzielec]

It didn't work

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[HomeBrew]

Was one of those lanes produced by your untagged primers?

[Adrian K]

In addition of what others had said, I would like to add in my idea.

If I were to get rid of the extra "larger" band, I personally would reduce further my annealing and extension time. This will not give "enough time" for Taq to have chance for larger amplification.

Try to reduce annealing time by 5-10seconds, and reduce extension time for 15 seconds. Or, you just use 10-15 seconds for your annealing time, and skip the extension time.

I do agree with Homebrew to reduce MgCl2 concentration, and reduce your buffer strength as well.
You can also try touchdown PCR.

Hope this help. Keep us update.
Adrian

Edited by adrian kohsf, 13 April 2010 - 10:48 PM.