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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*Sonication: 8 cycles of 15 seconds on, 90 seconds off, High Power | *Sonication: 8 cycles of 15 seconds on, 90 seconds off, High Power | ||
[[Image:Greenbuls1shearedgels.png|500px]]<br> | [[Image:Greenbuls1shearedgels.png|500px]]<br> | ||
Do have have gel image for 2nd shearing attempt | Do not have have gel image for 2nd shearing attempt (4 additional cycles), but looked better than first. Because final attempt with additional 2 cycles did not look right (more large fragments), decided to continue with subsequent steps to avoid further sonication problems. | ||
*End Repair: NEBENDRP on John Block B | *End Repair: NEBENDRP on John Block B | ||
*P2 Ligation: NEBLIGAT on John Block B | *P2 Ligation: NEBLIGAT on John Block B | ||
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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> | ||
*Clean up: 45 μL beads eluted in 30 μL LowTE | |||
*PicoGreen: 8.44 ng/μL | *PicoGreen: 8.44 ng/μL | ||
==Re-prep Greenbuls Plate 2== | |||
Used same SbfI-HF and ligase from previous library. | |||
*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 | |||
==Re-prep Sunbirds Plate 2 and Greenbuls Plate 2== | |||
Re-prep from existing plates. | |||
Received new SbfI-HF from NEB. | |||
*Digestion 2014-08-13 | |||
**Sunbirds: RADIGEST on Sork | |||
**Greenbuls: RADIGEST on Tony | |||
*Ligation 2014-08-13 | |||
**Sunbirds: Used Berkeley adapter plate and old T4 ligase. RADLIG on Sork | |||
**Greenbuls: Used mostly Miller adapter plate and new T4 ligase. RADLIG on Tony | |||
*Bead Cleanup 2014-08-13 | |||
*Sonication 2014-08-14 | |||
**8 cycles followed by 4 + 2 + 2 additional cycles (16 total) | |||
*Blunt End Repair 2014-08-14: NEBENDRP on John Block B | |||
*P2 Adapter Ligation 2014-08-14: NEBLIGAT on John Block B. Used new tube of P2. | |||
*Size Selection 2014-08-15: 40 μL + 25 μL beads | |||
*PCR 2014-08-15: NEBPCR on John Block B | |||
[[Image:Reprep-OSBLGB2-pcr.JPG|300px]] | |||
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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