I would like to capture specific cDNA/DNA and noticed that there are many protocols using different hyrbid system( different NaCl concentration, different detergent and additives) and different types of probes( DNA, RNA or LNA). I wonder which works best.
I have tried using RNA probes with 4×Denhardt's；4mM EDTA；0.1% SDS；0.05% Tween20；27ul DNA，2.5ug Cot-1，25ug Salmon Sperm DNA，500ng probe, 200pmol block RNA
and hybrid for 66 hours at 65oC, followed by binding to myone C1 dynabeads for 30min at room temperature. However, I get nothing.
(I followed protocol largely from the paper that's attached below)
Is there anybody providing any suggestions? Thank you in advance!
Article Pulling out the 1%: Whole-Genome Capture for the Targeted En...
Hi Yan,
Let me know if you got this procedure to work. Our procedure is similar. I have also made biotinylated RNA probes from cDNA clones and T7-tagged PCR products and it works great. One thing that is very important is how you amplify post-capture. We have found that for standard Illumina libraries, KAPA HiFi polymerase is the best (it comes in a 2X mastermix). NEB's Q5 I have heard is also equally good, but I have not tried it. Also check to see that for high GC targets that you reduce your insert size, as even with these polymerases, you will have regions sequence poorly even though in bulk the post-capture libraries appear to amplify well.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111756
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