vanu

Dear all,

I´m writing once more as I have not still finished obtaining my PCR products for sequencing. Now, that I have obtained enough purified PCR product, when I have run on gel my amplicons in order to observe if there is a unique band, prior to the pooling, I have observed that there are some bands which are bigger than my product of interest that is 600bp more or less.

As I indicated with the aim of getting enough purified material, I performed the next steps:
1. First I have done PCR with my primers and DNA total amount of 50 ng
2. I have run on gel the PCR product
3. I have gel purified my band of interest
4. I have used purified DNA as template and performed a second PCR
5. I have purified PCR product with PCR purification Kit

This is what I obtain (image 1), every band has been obtained using different pair of primers and DNA.

Does anyone know why do I get those bands larger than my product?
This DNA would be for sequencing, could those bands interfere in the analisis? And finally, does anyone know how can I improve my product without starting again from 0?

Thank you very much for your help in advance,

Thanks.

Hobglobin, thank you for the ideas, but I have already tried to optimize as much as possible my PCR reactions and can´t improve them more.

As Trof said, I will ask again the enterprise whether they need such amount of DNA. But, in my opinion even if 10-15 ng could be enough for sequencing, they won´t agree, as they have insisted with these conditions and on the other hand, also have asked for a gel image of the pool, and this 10 ng, I´m not sure wilI be enough to obtain a gel image...

Anyway, Trof, we have thought to perform a second PCR reaction, using DNA purified from gel with the same PCR conditions, in order to obtain a second PCR product but this time it is expected to obtain a clear unique band as we have used purified DNA, so then we would be able to purify it not from gel, but directly from the PCR product. What do you think about that? I think is a good idea but I´m not sure it will work...

But for sequencing they are asking me a minimun quantity of 1.5 ug and 20 ul...
so even if I increase a little bit more the concentration it wouldn´t be enough. What about if I perform a PCR of the PCR product?

Hi,

I just wanted to tell you that I have finally obtained my band of interest!So thank you all!
But now, that I have solved my problem with PCR reaction, another problem has come to me...even if I could perfectlly observed by band, there are also some inespecifities so, in order to purify the PCR product for posterior sequencing, I should do it performing a band purification from agarose gel, However I have already try it several times and have only obtained DNA concentrations ranging 10- 20 ng/ul, can anyone help me with this?

PD: I use QUIaquick Gel Extraction Kit, and have made a 0.8% agarose gel, also, I have changed a little bit the instructions indicated by the suppliers, as heating till 60 degrees the elution buffer...but nothing has improved the yield. Any suggestions?

Thank you very much in advance

Thanks a lot mdfenko, this will be really helpful!