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-   -   solexa adapter ligation question (http://seqanswers.com/forums/showthread.php?t=1292)

seqgirl123 03-01-2009 07:21 PM

solexa adapter ligation question
 
I am following the Solexa genomic DNA library prep protocol. I ran a gel after I sheared at 250bp, end repaired, 3'A extended, and ligated the genomic DNA to the adapters. I ran a gel on the ligated material in Lane 1. I also ran a gel on the DNA where the same things were done but I eliminated the 3'A extension step in Lane 2. Lane 3 contains DNA ladder.

Lane 1 material gave a band/smear around 250bp. Lane 2 material showed high molecular weight DNA.

If I understood how the adapters work from Solexa correctly, the adapters are double stranded and there is a T on one strand (3' end) of the adapter and the other strand (5') is phosphorylated? So, the 3'T on the adapter will ligate to the 3'A on the DNA and the 5' phosphorylated end of the adapter will ligate to the 5'phosphate on the blunt ended DNA?

And what I'm seeing in the gel in Lane 1 is right because the adapters cannot ligate together due to the incompatibility on their ends. They have Lane 2 shows DNA reannealed to itself and cause the high MW product.

Does this all sound right?

If yes and the 5' end of the adapter is phosphorylated, why doesn't this anneal to itself? Or where is the adapter phosphorylated if at all??

EPICENTRE 03-04-2009 07:07 AM

My understanding is that they use a standard A-tailing. The genomic fragments have 3'-A tails. The adaptors have a 5'-T overhang, not a 3'. This way you should get slightly improved ligation due to the 1-bp annealing in the ligation rxn. The overhangs also will prevent concatemers from forming.

Nihilo 10-13-2009 11:08 AM

My question might be naive, but the adapters should be able to ligate to either end of the DNA, right ? If this is the case, you would end up with some products with the same primer at each end ...and these products cannot be sequenced since the sequencing primer is specific to only one adapter ... am I missing something ?


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