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Old 01-05-2012, 12:18 AM   #1
visserm
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Location: South Africa

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Default 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
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Old 09-25-2013, 07:28 AM   #2
JenMar
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Default 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
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Old 09-25-2013, 11:48 PM   #3
visserm
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Location: South Africa

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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
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Old 09-26-2013, 08:36 AM   #4
JenMar
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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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