CTR:Notebook/PIRE/2014/07/09: Difference between revisions
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*New DNA plate prepped 2014-08-05 | *New DNA plate prepped 2014-08-05 | ||
*Digestion: RADIGEST on Tony 2014-08-06 | *Digestion: RADIGEST on Tony 2014-08-06 | ||
*Ligation: | *Ligation: RADLIG on Tony 2014-08-06 | ||
**Adapters from original Berkeley plate | **Adapters from original Berkeley plate | ||
*Clean up 2014-08-06 | *Clean up 2014-08-06 | ||
2014-08-07: | |||
*Sonication | |||
[[Image:Reprep-greenbuls2-sheared.JPG|200px]] | [[Image:Reprep-greenbuls2-sheared.JPG|200px]] | ||
8 cycles of 15 seconds on, 90 seconds off, high power. 2μL of ladder and sheared DNA. | 8 cycles of 15 seconds on, 90 seconds off, high power. 2μL of ladder and sheared DNA. | ||
*Blunt End Repair: | *Blunt End Repair: NEBENDRP on John Block B | ||
*P2 Adapter Ligation: NEBLIGAT on John Block B | |||
*Size selection: 40μL to remove large fragments, 25μL to remove small fragments | |||
*PCR: NEBPCR on John Block B | |||
[[Image:Reprep-greenbuls2-pcr.JPG|300px]] 1) 1μL template. 2) 2μL ladder. 3) 5μL PCR product | |||
→No amplification | |||
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Revision as of 15:27, 7 August 2014
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RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2
2μL of sheared DNA in each well
Troubleshooting
1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA
1) 1μL template, 2) 5μL PCR product
1&2) Sunbirds Plate 1, 3&4) Greenbuls Plate 1 (from re-sheared template) Re-prep Greenbuls Plate 1 using leftover DNA from plate2014-07-31:
2014-08-01:
1μL template, 2μL 100bp ladder, 5μL PCR product
Re-prep Greenbuls Plate 2
2014-08-07:
8 cycles of 15 seconds on, 90 seconds off, high power. 2μL of ladder and sheared DNA.
1) 1μL template. 2) 2μL ladder. 3) 5μL PCR product →No amplification
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