CTR:Notebook/PIRE/2014/07/09: Difference between revisions
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==Re-prep Greenbuls Plate 1 using leftover DNA from plate== | ==Re-prep Greenbuls Plate 1 using leftover DNA from plate== | ||
Used new SbfI-HF tube and new T4 ligase. | |||
2014-07-31: | 2014-07-31: | ||
*Digestion: RADIGEST on Tony | *Digestion: RADIGEST on Tony | ||
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==Re-prep Greenbuls Plate 2== | ==Re-prep Greenbuls Plate 2== | ||
Used same SbfI-HF and ligase from previous library. | |||
*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 | ||
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==Re-prep Sunbirds Plate 2 and Greenbuls Plate 2== | ==Re-prep Sunbirds Plate 2 and Greenbuls Plate 2== | ||
Re-prep from existing plates. | |||
Received new SbfI-HF from NEB. | Received new SbfI-HF from NEB. | ||
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*PCR 2014-08-15: NEBPCR on John Block B | *PCR 2014-08-15: NEBPCR on John Block B | ||
[[Image:Reprep-OSBLGB2-pcr.JPG|300px]] | [[Image:Reprep-OSBLGB2-pcr.JPG|300px]] | ||
*Clean up | 1) 1 μL Sunbird plate 2 template; | ||
2) 5 μL Sunbird plate 2 PCR product; | |||
3) 2 μL 100bp ladder; | |||
4) 1 μL Greenbul plate 2 template; | |||
5) 5 μL Greenbul plate 2 template | |||
→Using new SbfI-HF seems to work. New enzyme and ligase is ideal! | |||
*Clean up 2014-08-19: 45μL beads eluted in 30μL LowTE | |||
*PicoGreen | |||
**Sunbirds Plate 2: 4.74 ng/μL | |||
**Greenbuls Plate 2: 7.79 ng/μL | |||
==BioAnalyzer results from all 3 libraries== | |||
1) Sunbirds Plate 2; 2) Greenbuls Plate 1; 3) Greenbuls Plate 2 | |||
[[Image:Bioanalyzer OS2LG12.png|450px]] | |||
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Revision as of 09:26, 27 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 plateUsed new SbfI-HF tube and new T4 ligase. 2014-07-31:
2014-08-01:
1μL template, 2μL 100bp ladder, 5μL PCR product
Re-prep Greenbuls Plate 2Used same SbfI-HF and ligase from previous library.
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 Re-prep Sunbirds Plate 2 and Greenbuls Plate 2Re-prep from existing plates. Received new SbfI-HF from NEB.
1) 1 μL Sunbird plate 2 template; 2) 5 μL Sunbird plate 2 PCR product; 3) 2 μL 100bp ladder; 4) 1 μL Greenbul plate 2 template; 5) 5 μL Greenbul plate 2 template →Using new SbfI-HF seems to work. New enzyme and ligase is ideal!
BioAnalyzer results from all 3 libraries1) Sunbirds Plate 2; 2) Greenbuls Plate 1; 3) Greenbuls Plate 2
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