CTR:Notebook/PIRE/2014/07/09: Difference between revisions
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*Size Selection: 40μL beads to remove large fragments (decreased amount to reduce loss of DNA), 25μL to remove small | *Size Selection: 40μL beads to remove large fragments (decreased amount to reduce loss of DNA), 25μL to remove small | ||
*PCR: NEBPCR on John Block B | *PCR: NEBPCR on John Block B | ||
[[Image:Reprep-greenbuls1-pcr.JPG| | [[Image:Reprep-greenbuls1-pcr.JPG|300px]] 1μL template, 2μL 100bp ladder, 5μL PCR product<br> | ||
→Successful amplification! | →Successful amplification! | ||
<br> | <br> | ||
*PicoGreen: 8.44 ng/μL | *PicoGreen: 8.44 ng/μL | ||
==Re-prep Greenbuls Plate 2== | |||
*New DNA plate prepped 2014-08-05 | |||
*Digestion: RADIGEST on Tony 2014-08-06 | |||
*Ligation: RADLIG on Tony 2014-08-06 | |||
**Adapters from original Berkeley plate | |||
*Clean up 2014-08-06 | |||
2014-08-07: | |||
*Sonication | |||
[[Image:Reprep-greenbuls2-sheared.JPG|200px]] | |||
8 cycles of 15 seconds on, 90 seconds off, high power. 2μL of ladder and sheared DNA. | |||
*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<br> | |||
→No amplification | |||
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Revision as of 15:28, 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
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