CTR:Notebook/PIRE/2015/01/14: Difference between revisions
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Sirena Lao (talk | contribs) (Autocreate 2015/01/14 Entry for CTR:Notebook/PIRE) |
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==RAD Library Prep - | ==RAD Library Prep - Skinks Plate 1== | ||
* 96 well plate of 50ng of DNA 10uL | * 96 well plate of 50ng of DNA 10uL | ||
* Digestion ( | * Digestion (2015-01-14) | ||
**Mix and add to each well: | **Mix and add to each well: | ||
***0.68 uL water | ***0.68 uL water | ||
***1.20 uL NEBuffer 4 | ***1.20 uL NEBuffer 4 | ||
***0.12 uL SbfI-HF | ***0.12 uL SbfI-HF | ||
**Incubation: | **Incubation (RADIGEST on TONY): | ||
***37 degrees for 60 minutes | ***37 degrees for 60 minutes | ||
***65 degrees for 20 minutes | ***65 degrees for 20 minutes | ||
* P1 adapter ligation ( | * P1 adapter ligation (2015-01-14) | ||
**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: | ||
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***0.16 uL ATP | ***0.16 uL ATP | ||
***0.16 uL T4 Ligase | ***0.16 uL T4 Ligase | ||
**Incubation: | **Incubation (RADLIG on TONY): | ||
*** 20 degrees for 60 minutes | *** 20 degrees for 60 minutes | ||
*** 65 degrees for 20 minutes | *** 65 degrees for 20 minutes | ||
* Clean up ( | * Clean up (2015-01-14) | ||
**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: | ||
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***Elute in 100 uL LowTE | ***Elute in 100 uL LowTE | ||
* Sonication ( | * Sonication (2015-01-1) | ||
**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:Sunbird1 sheared 2014-05-01.JPG|200px]] | [[Image:Sunbird1 sheared 2014-05-01.JPG|200px]] | ||
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3) 3uL sheared DNA | 3) 3uL sheared DNA | ||
*Blunt End Repair ( | *Blunt End Repair (2015-01-1) | ||
**Mix: | **Mix: | ||
***50.0 uL fragmented DNA | ***50.0 uL fragmented DNA | ||
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***65 degrees for 30 minutes | ***65 degrees for 30 minutes | ||
*P2 adapter ligation ( | *P2 adapter ligation (2015-01-1) | ||
**Add to mix: | **Add to mix: | ||
***15.0 uL Blunt/TA Ligase Master Mix | ***15.0 uL Blunt/TA Ligase Master Mix | ||
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***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection ( | *Size selection (2015-01-) | ||
**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: | ||
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***Elute in 20 uL LowTE | ***Elute in 20 uL LowTE | ||
*PCR amplification ( | *PCR amplification (2015-01-1) | ||
**Mix: | **Mix: | ||
***5 uL DNA | ***5 uL DNA | ||
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1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product | 1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product | ||
*Bead clean up ( | *Bead clean up (2015-01-1) | ||
**Use 45 uL AMPure beads (1:1) | **Use 45 uL AMPure beads (1:1) | ||
**2 washes of 200 uL 80% EtOH | **2 washes of 200 uL 80% EtOH | ||
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[[Image:Sunbirds1 bioanalyzer.jpg|450px]] | [[Image:Sunbirds1 bioanalyzer.jpg|450px]] | ||
*Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) ( | *Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2015-0) | ||
Revision as of 12:06, 14 January 2015
PIRE RAD Library Preps | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
RAD Library Prep - Skinks Plate 1
1) 1uL pre-sheared DNA, 2) 2uL 100bp low scale ladder, 3) 3uL sheared DNA
1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product
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