SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   Sample Prep / Library Generation (http://seqanswers.com/forums/forumdisplay.php?f=25)
-   -   DNase treatment for SMART Seq2 (http://seqanswers.com/forums/showthread.php?t=77457)

candida utilis 08-07-2017 08:44 AM

DNase treatment for SMART Seq2
 
I notice that DNase I treatment is not included in the SMART Seq2 protocol. May I know how efficient Oligo-dT30VN will bind to mRNA only? Is there any possibility that Oligo-dT30VN can also amplify DNA from genomic DNA, if there is no DNase I treatment?

Thanks

cmbetts 08-07-2017 10:33 AM

There's a couple reasons not to worry about it, but the primary ones are:
1) The denaturation temperature before RT isn't high enough to melt high MW gDNA
2) Even if gDNA was primed, the template switching mechanism requires the RT to hit a 5' end and neither the RT nor the PCR polymerase are anywhere processive enough to worry about capturing whole chromosomes.

jteeee2 08-08-2017 09:37 AM

If you're really interested DNase treating, we have had good success incorporating this product into the front end of several RNA Seq protocols (including SmartSeq). http://arcticzymes.com/technology/kits/heat-and-run/

We have seen that significant DNA contamination can affect the quality of the SmartSeq library, so I think it's definitely worth considering.


All times are GMT -8. The time now is 06:40 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.