10 replies to this topic

[Tina Hansen]

HI

I have experienced some weird results from my sequencing lately.

I'm trying to sequence inserts in pCR2.1-Topo vector using the vector primers supplied within the kit (M13 forward (-20) and M13 reverse).

Prior to growing Over Night (ON) cultures on the clones, I test using the vector primers that the clone indeed does contain an insert. This PCR works fine and I can pick the clones that appear to have an insert of correct size. (The fragments a want to clone, may vary in size and I need a clone in a certain size).

After purifying plasmid from ON cultures, I sequence the clones to pick a clone with the correct sequence. I have done this many times before, but not by using the vector primers. Here is the problem:

1. It appears that the company has mixed up the primers, because I get the reverse primer's sequence with the forward primer. This I can see because after a few hundred bases the forward primer's sequence pops up. If it really was the forward primer that was used it should have been the reverse primer's sequence popping up. FYI it is not me mixing up the primers, I have done the experiment twice Has anyone else experienced this mix up?

2. One of the primers NEVER work in the DNA sequencing. Taken into account the potential primer "mix-up" it is the M13 Forward primer that never works. This is strange to me since the to primers as a pair function fine in the PCR. Does anyone have an explanation to why the primer is not working when used for DNA sequencing?

I have attached the chromatograms for both primers for one of the clones, so that you can see how it looks when I don't get a sequence for the forward primer.

Thanks in advance

Tina

Attached Thumbnails

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Attached Files

•  KK002085a.bmp   55.99K   66 downloads

[Ameya P]

Tina Hansen, on 11 January 2011 - 11:43 PM, said:

2. One of the primers NEVER work in the DNA sequencing. Taken into account the potential primer "mix-up" it is the M13 Forward primer that never works. This is strange to me since the to primers as a pair function fine in the PCR. Does anyone have an explanation to why the primer is not working when used for DNA sequencing?

I am not sure, if there are mix-up's with primers from a kit (the company must be be having some QC checks), but PCR can work with a single primer. So to add fuel to your theory, it could be that your PCR is working just because of the single 'working primer' and that could also explain why you do not get any specific read out of your sequencing....

[Tina Hansen]

That could definetly explain the phenomenon. But would a single primer PCR not generate a smear rather than a distinct band? See attached file.

Attached Files

•  KK002085a.bmp   55.99K   78 downloads

[seanspotatobusiness]

I don't understand. How could you produce detectable amounts of DNA with only one working primer?

[mdfenko]

primers that work for pcr don't always work with sequencing. you should design a new primer to replace the one that doesn't work.

[philman]

mdfenko, on 12 January 2011 - 07:09 AM, said:

primers that work for pcr don't always work with sequencing. you should design a new primer to replace the one that doesn't work.

Indeed, if a primer binds to 2 places on the DNA, but one of those binding sites is the wrong side of the other primer, or too far away, then the amount of DNA made from that primer will be tiny compared to the amount made by the two primers binding to the correct sites. But in sequencing, if they bind more than one site it will screw the whole thing up.

[Ameya P]

seanspotatobusiness, on 12 January 2011 - 06:36 AM, said:

I don't understand. How could you produce detectable amounts of DNA with only one working primer?

Well, this is what we do for RAPD as well.

[Ameya P]

Tina Hansen, on 12 January 2011 - 05:41 AM, said:

That could definetly explain the phenomenon. But would a single primer PCR not generate a smear rather than a distinct band? See attached file.

(I tried to multi-quote, but it did not work ). So again, its not necessary that single primer will generate a smear alone. If the extension is uniform, it will give a distinct band.

[ElHo]

RAPD usually uses short primers (~ 4 NTs) that will bind to different locations in the genome, thereby leading to pcr products. M13 sequencing primers are much longer and therefore much more unlikely to frequently anneal at different positions, especially when using plasmid DNA as template. So I don't think that you get a pcr product with only one primer working in your case. But you could easily test that by setting up a pcr with only one single primer. I agree with mdfenko and philman that one of your primers might work well in pcr in combination with the other one but not in sequencing.

[Tina Hansen]

Thank you all for your responses.

I have solved the problem with the "primer mix-up". Apperently, when looking at the topo-vector, the company has named the up-stream primer of the cloning site reverse and the down-stream primer for forward. That was what confused me. So there where no mix-up after-all, just difference in how we define a forward and reverse primer.

I haven't solved the mystery of why one primer is not working in Sequencing. I will however change primer batch and start with fresh reagents and hope this will solve the issue.

Thanks again

Tina

[phage434]

Topo cloning is not directional, so the "forward" direction is aligned with your idea of forward only half the time.  You picked a clone with the orientation different from your expectation.

A PCR could work fine with primers that bind to both a template for amplification, and (separately) to a vector region.  If you insert the PCR product into that vector, then there will be two copies of the sequence in the resulting plasmid.  You will not get interpretable sequencing results using that primer.