Lets me explain my experimental strategy. I would like to characterise the integration site of HIV in my cell type and to do that i plan to extract DNA, sonicate them (~300 to 500bp), use End-It™ DNA End-Repair Kit to repair the fragment, use NEBNext dA-Tailing Module to add single dA to 3' END and than link a partially double stranded linker with one nucleotide 3’ T overhang to ligate to the genomic DNA fragments. Amplified all of my bank with primer on the flanking sequence, than amplified my stecific region of interrest with a primer on the flanking region and a other on the LTR of my virus. and than i would like to add the adapter and sequence the remaining fragments.
So my questions are multiple :
First -> should I purified DNA after the repairing kit to avoid the T4 pnK ? or should i just purified DNA after the dA tailing as it's preconize ?
Second -> For the synthesizing of the partially double stranded linker how can i processed ? should i make than produce separatly (each strand ?) and how can i have one nucleotide 3’ T ?
Last -> for the ligation should i do it directly after the daTailing ? or after the purification ? and should i do a other purification dirtectly after the ligation ?
Thank you very much
I am not sure you need to do sonication and adaptor ligation at all. Why not digest the DNA with an enzyme that cuts in your viral template at known positions. Then dilute the DNA and ligate it so that the DNA circularizes. Using primers for your viral genome at the ends of the genome, maybe the LTRs, you can then do PCR and amplify the seances at the junctions which read in tot eh cell genome. These PCR products can then be sent for sequencing.
The workflow you are using for your experimental strategie is similar to the initial steps of the workflow i use for methylome library preparation, we purify by column or using AMPure XP beads after Endit Repair, dA tailing and Adapter ligation.
http://www.nature.com/nprot/journal/v10/n3/full/nprot.2014.114.html
For the adapter synthesis we usually design each strand and we prepare the duplex adapter by annealing both oligonucleotides this is sometimes a little tricky if you do not have any experience annealing primers to obtain duplex adapter, also do not forget to add add 5’-phosphates to the strand ends because these are important when you are performing a ligation reaction. Oligo synthesis companies like IDT do the annealing and 5’-phosphates at request if you want to go the easiest way.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics