CTR:Notebook/PIRE/Entry Base: Difference between revisions
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Sirena Lao (talk | contribs) |
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(7 intermediate revisions by the same user not shown) | |||
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* Clean up (2014-04-30) | * Clean up (2014-04-30) | ||
**Take 5 uL | **Take 5 uL from each well and combine to 1.7 mL tube | ||
**AMPure bead clean up: | **AMPure bead clean up: | ||
***Use 480 uL beads to DNA (1:1) | ***Use 480 uL beads to DNA (1:1) | ||
Line 61: | Line 61: | ||
***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection | *Size selection (2014-05-01) | ||
**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: | ||
Line 69: | Line 69: | ||
***Elute in 20 uL LowTE | ***Elute in 20 uL LowTE | ||
*PCR amplification (2014-05-01) | |||
**Mix: | |||
***5 uL DNA | |||
***25 uL NEBNext High Fidelity 2x PCR Master Mix | |||
***18 uL water | |||
*** 1 uL P1 adapter primer (25 uM) | |||
*** 1 uL P2 adapter primer (25 uM) | |||
**PCR cycle: | |||
***98 degrees for 30 seconds | |||
***15 cycles of: | |||
****98 degrees for 10 seconds | |||
****65 degrees for 30 seconds | |||
****72 degrees for 30 seconds | |||
***72 degrees for 5 minutes | |||
[[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]] | |||
1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product | |||
*Bead clean up (2014-05-02) | |||
**Use 45 uL AMPure beads (1:1) | |||
**2 washes of 200 uL 80% EtOH | |||
**Elute in 30 uL LowTE | |||
*Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip | |||
[[Image:Sunbirds1 bioanalyzer.jpg|450px]] | |||
*Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2014-05-05) | |||
==RAD Library Prep - == | |||
* | |||
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Revision as of 16:36, 5 May 2014
PIRE RAD Library Preps | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
RAD Library Prep - Olive Sunbirds 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
RAD Library Prep -
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