Explain me End Repair
Posted 16 June 2010 - 02:41 PM
Is there a book or a website that explains CLEARLY what each enzyme is doing and why we need them?
Also, if I did a linear amplification (one primer only) and wanted to cut all the overhangs after that, which enzyme should I use?
I'd highly appreciate your help.
Posted 17 June 2010 - 06:43 AM
The T4 PNK step phosphorylates the 5' ends of the blunt-ended DNA, to enable subsequent ligation of adaptors.
A better approach to library preparation, though, is to avoid shearing, end-repair, and ligation altogether.
For your second question, do you mean you are doing a linear amplification of genomic DNA, and then using the amplification products for library prep?
Edited by epibio, 17 June 2010 - 06:44 AM.
Posted 17 June 2010 - 07:56 AM
I do not shear my DNA because I am working with very degraded DNA from bones, but why do you say I should avoid the end repair and ligation? How else could I add my adaptors?
You are correct, I would like to use the PCR products + the rest of the extract actually for the library. My problem is that in my extracts, I have very small amounts of degraded human DNA and tons of microbial DNA. I was hoping that several cycles of linear amplifications targeting the human DNA would help increase and create DNA having a better quality, which would ligate the adaptors better. Problem is I may end up with very long fragments of ssDNA on the 5' end. Could Klenow deal with that or is it usually used to fill up small gaps?
Posted 17 June 2010 - 08:35 AM
Anyway, my original comment about avoiding shearing/end-repair/adaptor ligation is that there are multiple steps involved, while the Nextera method combines those into a single reaction. It's probably not suitable for degraded DNA, though.
Klenow is typically used to fill in small overhangs, but it should still work in your case.
Posted 17 June 2010 - 09:11 AM
That's exactly what I am doing ..or planning on doing. I noticed that degraded DNA does't do good with library prep so I was hoping to enrich first (and then again later after amplifying the library)
I'll have to do each step at a time.
Posted 17 June 2010 - 10:12 AM
One hour at 37C should be more than sufficient, but I'd suggest using the exo minus mutant rather than "standard" Klenow.
Posted 17 June 2010 - 11:51 AM
Posted 17 June 2010 - 12:22 PM
Posted 17 June 2010 - 01:02 PM
is that the baby I need?
Edited by Maddie, 17 June 2010 - 01:15 PM.
Posted 17 June 2010 - 01:30 PM
Posted 21 June 2010 - 11:30 AM
Does anyone has experience with the preCR kit of NEB?