question on solexa genomic dna libraries

[seqgirl123]

Appreciate any help on this.

I am following the Genomic Dna Library protocol from Solexa. In the final step (step 6) which amplifies the adapter enriched products from the gel by pcr, Solexa uses Phusion Dna Polymerase. For some reason, this step does not always work for me as I do not get any pcr bands in my region (250bp). I did the first few times with my control, but it does not always work with my actual samples. I will increase my cycles from 18 to 22 and see what that does, but are there alternatives I can try to the Phusion DNA polymerase? Will any DNA polymerase suffice? I was thinking of using the Taq DNA polymerase I use in my other PCR (non-solexa related) and I know works. Have others have tried using polymerases besides the Phusion that solexa provides?

[ScottC]

I think any high-fidelity enzyme should be OK. But I think the reason is probably not the polymerase. It should work. Are you recovering enough DNA from the size-selection step?

Scott.

[seqgirl123]

By size selection step, do you mean the gel that is run in step 5?

Lately, I have been running a gel after every step to confirm DNA has sheared properly, ligated properly etc. These gels look fine in that they all show DNA material in each lane or a specific bp range so I can only see it is something in the last step.

I don't know if it makes sense to run a gel right before I do the PCR as I have not done that. Will this gel confirm if I have recovered enough DNA material for the PCR step?

[ScottC]

Yes, I mean the gel extraction step.

Do you have access to a small-volume spectrophotometer like a Nanodrop, or a fluorimeter like a Quant-iT for example?

Do you see anything on your gel after the PCR?

[dolaimi]

I guess there may be some problem in end repair and adapter ligation. If the adapter were not able to be ligated with your DNA fragments effeciently, you would not get enough PCR product.

[seqgirl123]

Ok, I will measure DNA concentration after gel extraction step and see what this shows me.

My other question is, I have been running a gel after each step. I see DNA material and confirms to me that my column spins are eluting DNA. I have also been trying to compare what each gel looks like in each step. Would running a gel after end repair and adapter ligation show that step has worked? Or what would give me a confirmation that each step has worked properly before I run the PCR?