CTR:Notebook/PIRE/2015/08/25: Difference between revisions
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***Elute in 100 uL LowTE | ***Elute in 100 uL LowTE | ||
* Sonication (2015-08-26) | * Sonication - edited to get smaller average fragments (2015-08-26) | ||
**BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power | **BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power | ||
[[Image:Sheared08262015 01.JPG|150px]] | [[Image:Sheared08262015 01.JPG|150px]] | ||
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***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection (2015-08-26) | *Size selection - edited to select for smaller insert (2015-08-26) | ||
**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: | ||
*** | ***55 uL beads to remove large fragments | ||
***25 uL beads to remove small fragments | ***25 uL beads to remove small fragments | ||
***3 washes of 200 uL 80% EtOH | ***3 washes of 200 uL 80% EtOH | ||
Line 87: | Line 87: | ||
****72 degrees for 30 seconds | ****72 degrees for 30 seconds | ||
***72 degrees for 5 minutes | ***72 degrees for 5 minutes | ||
[[Image: | **Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel. | ||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product | [[Image:PCRproduct08272015.JPG|200px]] | ||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br> | |||
→Visible amplification, but size range difficult to discern. | |||
*Re-run PCR to improve visibility and get more amplification (2015-08-27) | |||
*Re-run | |||
**Mix: | **Mix: | ||
***10 uL DNA | ***10 uL DNA | ||
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*** 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) | ||
1) 2 uL | |||
*PicoGreen: | <br><br> | ||
--------------------------------------------- | |||
<br> | |||
Steps below have not yet been completed. | |||
<br> | |||
*Bead clean up (2015-) | |||
**Use 45 uL AMPure beads (1:1) | |||
**2 washes of 200 uL 80% EtOH | |||
**Elute in 30 uL LowTE | |||
*Quant DNA library using PicoGreen: 1.53 ng/μL. | |||
<br><br> | <br><br> | ||
*Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015- | *Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-) | ||
[[Image:Bioanalyzer-skinks-plate1.jpg|450px]] | [[Image:Bioanalyzer-skinks-plate1.jpg|450px]] | ||
*Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 | *Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 PE) (2015-) | ||
Revision as of 12:04, 27 August 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 3 (48 wells, Reruns)
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product →Visible amplification, but size range difficult to discern.
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