SOLiD Mate Pair Library Prep

busypops · 01-17-2011, 04:52 AM

Has anyone tried altering the Nick Translation reaction time in the SOLiD mate pair library protocol? I need a final library sixe of around 400bp so think I need to alter the nick translation time. I ried 8 minutes and got 300bp, anyone else tried altering the time and if so could you share your experiemnces?
Thanks in advance

pmiguel · 01-18-2011, 07:05 AM

Hey, you posted this in the Illumina forum. You want the Solid forum. (Maybe a moderator would be so kind as to move this thread to the solid forum?)

We actually use 3 different nick translation times, then pool them before the gel isolation step. We are hoping that will reduce systematic bias a little. That is, if there are sequence contexts where the nick translating polymerase is very slow or very fast, they might be missed with one time point because the resulting libraries will be too larger or too small, respectively. But by using 3 time points we might move the too slow or too fast pools into the correct size window.

We just re-made a library from a species that we knew had DNA that was a slow nick-translator. 10, 12 and 14 minutes were what we used for it. That gave us a smear from about 150-350 bp on the gel. We took stuff above 250 bp.

Any particular reason you are going for 400 bp instead of the recommended 300 bp?

--
Phillip

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01-17-2011, 04:52 AM	#1
busypops Junior Member Location: Edinburgh Join Date: Jan 2011 Posts: 5	SOLiD Mate Pair Library Prep Has anyone tried altering the Nick Translation reaction time in the SOLiD mate pair library protocol? I need a final library sixe of around 400bp so think I need to alter the nick translation time. I ried 8 minutes and got 300bp, anyone else tried altering the time and if so could you share your experiemnces? Thanks in advance

01-18-2011, 07:05 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Hey, you posted this in the Illumina forum. You want the Solid forum. (Maybe a moderator would be so kind as to move this thread to the solid forum?) We actually use 3 different nick translation times, then pool them before the gel isolation step. We are hoping that will reduce systematic bias a little. That is, if there are sequence contexts where the nick translating polymerase is very slow or very fast, they might be missed with one time point because the resulting libraries will be too larger or too small, respectively. But by using 3 time points we might move the too slow or too fast pools into the correct size window. We just re-made a library from a species that we knew had DNA that was a slow nick-translator. 10, 12 and 14 minutes were what we used for it. That gave us a smear from about 150-350 bp on the gel. We took stuff above 250 bp. Any particular reason you are going for 400 bp instead of the recommended 300 bp? -- Phillip