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-   -   PippinPrep for TruSeq small RNA library selection (http://seqanswers.com/forums/showthread.php?t=16622)

visserm 01-05-2012 12:18 AM

PippinPrep for TruSeq small RNA library selection
 
Hi all,

I want to use the PippinPrep for size selection of my TruSeq small RNA libraries. In the TruSeq kit they give the specs for selection an a polyacrylamide gel. They however compensate (according to Illumina) for a ~7 nt deviation in size from what is expected for adapters adding 118 nt. According to them a 21 nt insert fragment will run at ~146 on a PAA gel. Is there somebody who has successfully used the PippinPrep (3% cassettes) and will be able so tell me with the settings they used to select the fragment containing the 21 and 24 nt inserts?

Thanks
Marike

JenMar 09-25-2013 07:28 AM

Pippen Prep and small RNA cleanup
 
Hi Marike,

Your post was made quite a long time ago so I hope that your have resolved the problem and can help me. I am currently trying to do the same thing. I set up the Pippen Prep using a broad range size selection setting the parameters to 110bp start and 160bp end. I pooled all my miRNA libraries before loading on the gel for a total load of about 1ug/lane. I ended up with a very nice sharp peak as viewed by the Tape Station, however the peak is at 119bp, which is quite a bit off from 144-150bp. I am hoping there is a shift between the peak that the Tape Station shows and what the Pippen Prep collected and that my end product is really the correct size. I am getting ready to run PAGE as the illumina protocol suggests and see where this peak I collected runs. Please let me know if you have any suggestions. I would love to use the Pippen Prep for cleanup.

Thanks,
JenMar

visserm 09-25-2013 11:48 PM

Hi JenMar

I haven't used the Pippin in a while and in the end started using PAGE (I do not have easy access to the machine anymore). In order to avoid selecting libraries without an insert I set the range from 133 to 156. This gave me the correct library size range without any adapter-dimers or primer-dimers. I remember that, on the Pippin, I got very sharp peak around 120 that I didn't collect, so the empty library were definitely there before size selection but not afterwards. A library without an insert is around 118 nt so I hope you didn't include the empty library, but PAGE should definitely clarify that and provide the opportunity to select for the actual library with insert.

All the best
Marike

JenMar 09-26-2013 08:36 AM

Hi Marike,

Thanks so much for getting back to me. I will try the range you suggested. We do have a Pippen in the lab so I would like to utilize it since there is not as much cleanup afterwards compared to the PAGE method.

Thanks again!
JenMar


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