Hey everyone,
I succeed in RRBS protocol following Nature protocol, using low amount of DNA. http://www.ncbi.nlm.nih.gov/pubmed/?...tion+profiling
My problems started when I tryed Barcode adapters for multiplexing (from Truseq Kit). I followed the same protocol just changing the adapters and primers. 2 things happened:
- I got this huge band adapter size. I diluted the primers 3x and it keeps happening.
- I tryed a different approach ( from other protocol) where I bisulfite converted the DNA, amplified it (6 cycles), then went to the gel size selection step , and finally did more 12/14 cycles. This way, I am able to cut the adapters away. But following this I got almost nothing after final PCR amplification.
- Not to mention the smear is shorter when compared to smear from no barcode adapters, even the gel cut being exactly the same.
Has anyone succeed using 5ng or less to multiplexing?
I succeed in RRBS protocol following Nature protocol, using low amount of DNA. http://www.ncbi.nlm.nih.gov/pubmed/?...tion+profiling
My problems started when I tryed Barcode adapters for multiplexing (from Truseq Kit). I followed the same protocol just changing the adapters and primers. 2 things happened:
- I got this huge band adapter size. I diluted the primers 3x and it keeps happening.
- I tryed a different approach ( from other protocol) where I bisulfite converted the DNA, amplified it (6 cycles), then went to the gel size selection step , and finally did more 12/14 cycles. This way, I am able to cut the adapters away. But following this I got almost nothing after final PCR amplification.
- Not to mention the smear is shorter when compared to smear from no barcode adapters, even the gel cut being exactly the same.
Has anyone succeed using 5ng or less to multiplexing?
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