CTR:Notebook/PIRE/2015/08/31: Difference between revisions
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*** 65 degrees for 20 minutes | *** 65 degrees for 20 minutes | ||
* Clean up (2015- | * Clean up (2015-09-01) | ||
**Take 10 uL from each well and combine to 1.7 mL tube | **Take 10 uL from each well and combine to 1.7 mL tube | ||
**AMPure bead clean up: | **AMPure bead clean up: | ||
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***Elute in 100 uL LowTE | ***Elute in 100 uL LowTE | ||
* Sonication - edited to get smaller average fragments (2015- | * Sonication - edited to get smaller average fragments (2015-09-01) | ||
**BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power | **BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power | ||
[[Image:Sheared08262015 01.JPG|150px]] | [[Image:Sheared08262015 01.JPG|150px]] | ||
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2) 2uL sheared DNA | 2) 2uL sheared DNA | ||
*Blunt End Repair (2015- | *Blunt End Repair (2015-09-01) | ||
**Mix: | **Mix: | ||
***50.0 uL fragmented DNA | ***50.0 uL fragmented DNA | ||
Line 52: | Line 52: | ||
***65 degrees for 30 minutes | ***65 degrees for 30 minutes | ||
*P2 adapter ligation (2015- | *P2 adapter ligation (2015-09-01) | ||
**Add to mix: | **Add to mix: | ||
***15.0 uL Blunt/TA Ligase Master Mix | ***15.0 uL Blunt/TA Ligase Master Mix | ||
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***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection - edited to select for smaller insert (2015- | *Size selection - edited to select for smaller insert (2015-09-01) | ||
**Add 16.5 uL water for a total of 100 uL | **Add 16.5 uL water for a total of 100 uL | ||
**AMPure bead size selection: | **AMPure bead size selection: | ||
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***Elute in 20 uL LowTE | ***Elute in 20 uL LowTE | ||
*PCR amplification (2015- | *PCR amplification (2015-09-02) | ||
**Mix: | **Mix: | ||
***5 uL DNA | ***5 uL DNA |
Revision as of 09:27, 31 August 2015
PIRE RAD Library Preps | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
RAD Library Prep - 48-sample libraries for Butterflies, Frogs, Mice, Skinks
1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product →Visible amplification, but size range difficult to discern.
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
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