CTR:Notebook/PIRE/2014/07/09: Difference between revisions
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==RAD Library Prep - Olive Sunbirds Plate 1== | ==RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2== | ||
* 96 well plate of 50ng of DNA 10uL | * 96 well plate of 50ng of DNA 10uL | ||
<br> | |||
* | <b>For Digestion & Ligation steps: | ||
*Sunbirds Plate 2 - 2014-07-09, Sork thermocycler | |||
*Greenbuls Plate 1 - 2014-07-11, Tony thermocycler | |||
*Greenbuls Plate 2 - 2014-07-14, Sork thermocycler</b> | |||
<br> | |||
* Digestion | |||
**Mix and add to each well: | **Mix and add to each well: | ||
***0.68 uL water | ***0.68 uL water | ||
Line 18: | Line 23: | ||
***65 degrees for 20 minutes | ***65 degrees for 20 minutes | ||
* P1 adapter ligation | * P1 adapter ligation | ||
**Add 2 uL indexed P1 adapter (10nM) to each well | **Add 2 uL indexed P1 adapter (10nM) to each well | ||
**Mix and add to each well: | **Mix and add to each well: | ||
Line 28: | Line 33: | ||
*** 20 degrees for 60 minutes | *** 20 degrees for 60 minutes | ||
*** 65 degrees for 20 minutes | *** 65 degrees for 20 minutes | ||
<br> | |||
<b>The rest of the steps were done with all 3 libraries:</b> | |||
* Clean up | * Clean up - 2014-07-15 | ||
**Take 5 uL from each well and combine to 1.7 mL tube | **Take 5 uL from each well and combine to 1.7 mL tube | ||
**AMPure bead clean up: | **AMPure bead clean up: | ||
***Use | ***Use ~460 uL beads to DNA (1:1) | ||
***2 washes of 800 uL 80% EtOH | ***2 washes of 800 uL 80% EtOH | ||
***Elute in 100 uL LowTE | ***Elute in 100 uL LowTE | ||
* Sonication | * Sonication - 2014-07-15 | ||
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power | **BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power | ||
[[Image: | [[Image:RADbirds-shearinggels.png|500px]] | ||
2μL of sheared DNA in each well | |||
<br> | |||
2014-07-16: | |||
<br> | |||
*Blunt End Repair | *Blunt End Repair | ||
**Mix: | **Mix: | ||
***50.0 uL fragmented DNA | ***50.0 uL fragmented DNA | ||
***5.5 uL | ***5.5 uL LowTE | ||
***3.0 uL End Prep Enzyme Mix | ***3.0 uL End Prep Enzyme Mix | ||
***6.5 uL End Repair Reaction Buffer | ***6.5 uL End Repair Reaction Buffer | ||
**Incubation: | **Incubation: on John Block B | ||
***20 degrees for 30 minutes | ***20 degrees for 30 minutes | ||
***65 degrees for 30 minutes | ***65 degrees for 30 minutes | ||
*P2 adapter ligation | *P2 adapter ligation | ||
**Add to mix: | **Add to mix: | ||
***15.0 uL Blunt/TA Ligase Master Mix | ***15.0 uL Blunt/TA Ligase Master Mix | ||
***2.5 uL P2 RAD adapter (5 uM) | ***2.5 uL P2 RAD adapter (5 uM) | ||
***1.0 uL Ligation Enhancer | ***1.0 uL Ligation Enhancer | ||
**Incubation: | **Incubation: on John Block B | ||
***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection | *Size selection | ||
**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: | ||
Line 69: | Line 76: | ||
***Elute in 20 uL LowTE | ***Elute in 20 uL LowTE | ||
*PCR amplification | *PCR amplification | ||
**Mix: | **Mix: | ||
***5 uL DNA | ***5 uL DNA | ||
Line 76: | Line 83: | ||
*** 1 uL P1 adapter primer (25 uM) | *** 1 uL P1 adapter primer (25 uM) | ||
*** 1 uL P2 adapter primer (25 uM) | *** 1 uL P2 adapter primer (25 uM) | ||
**PCR cycle: | **PCR cycle: on John Block B | ||
***98 degrees for 30 seconds | ***98 degrees for 30 seconds | ||
***15 cycles of: | ***15 cycles of: | ||
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****72 degrees for 30 seconds | ****72 degrees for 30 seconds | ||
***72 degrees for 5 minutes | ***72 degrees for 5 minutes | ||
[[Image: | [[Image:RADbirds-PCRs.png|400px]]<br> | ||
1) 1 | 1) 1 μL Sunbird Plate 2 template, 2) 5 μL Sunbird Plate 2 PCR product<br> | ||
3) 1 μL Greenbul Plate 1 template, 4) 5 μL Greenbul Plate 1 PCR product<br> | |||
5) 1 μL Greenbul Plate 2 template, 6) 5 μL Greenbul Plate 2 PCR product<br> | |||
<br> | |||
→ PCR products do not look 2x as bright as the template. No significant amplication. | |||
<br> | |||
==Troubleshooting== | |||
*Tried re-prep of leftover sheared DNA (Blunt End Repair → PCR), but no difference | |||
<br> | |||
*Used old P1 primer in amplification | |||
[[Image:PCR-oldP1primer.JPG|300px]]<br> | |||
→ Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying. | |||
<br> | |||
*Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments. | |||
[[Image:Sheared-greenbuls2.JPG|300px]] | |||
1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA<br> | |||
*1st shearing step looks good - proceed to Blunt End Repair → PCR | |||
[[Image:PCR-greenbuls2.JPG|300px]] | |||
1) 1μL template, 2) 5μL PCR product<br> | |||
→ Still no amplification. Sonication is not the issue. | |||
<br> | |||
*Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer | |||
[[Image:Goodtemplate P2test.JPG|300px]] | |||
1&2) Sunbirds Plate 1, 3&4) Greenbuls Plate 1 (from re-sheared template)<br> | |||
→ PCR with good template still works. P2 primer only does not work at all | |||
==Re-prep Greenbuls Plate 1 using leftover DNA from plate== | |||
Used new SbfI-HF tube and new T4 ligase. | |||
2014-07-31: | |||
*Digestion: RADIGEST on Tony | |||
*Ligation: RADLIG on Tony | |||
**Used adapters from original Berkeley plate (undiluted) | |||
*Clean up | |||
*Sonication: 8 cycles of 15 seconds on, 90 seconds off, High Power | |||
[[Image:Greenbuls1shearedgels.png|500px]]<br> | |||
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 | |||
*P2 Ligation: NEBLIGAT on John Block B | |||
<br> | |||
2014-08-01: | |||
*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 | |||
[[Image:Reprep-greenbuls1-pcr.JPG|300px]] 1μL template, 2μL 100bp ladder, 5μL PCR product<br> | |||
→Successful amplification! | |||
<br> | |||
*Clean up: 45 μL beads eluted in 30 μL LowTE | |||
*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 | ||
[[Image: | **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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