CTR:Notebook/PIRE/Entry Base: Difference between revisions
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== | ==RAD Library Prep - Olive Sunbirds Plate 1== | ||
* | * 96 well plate of 50ng of DNA 10uL | ||
* Digestion (2014-04-30) | |||
**Mix and add to each well: | |||
***0.68 uL water | |||
***1.20 uL NEBuffer 4 | |||
***0.12 uL SbfI-HF | |||
**Incubation: | |||
***37 degrees for 60 minutes | |||
***65 degrees for 20 minutes | |||
* P1 adapter ligation (2014-04-30) | |||
**Add 2 uL indexed P1 adapter (10nM) to each well | |||
**Mix and add to each well: | |||
***1.28 uL water | |||
***0.40 uL NEBuffer 4 | |||
***0.16 uL ATP | |||
***0.16 uL T4 Ligase | |||
**Incubation: | |||
*** 20 degrees for 60 minutes | |||
*** 65 degrees for 20 minutes | |||
* Clean up (2014-04-30) | |||
**Take 5 uL from each well and combine to 1.7 mL tube | |||
**AMPure bead clean up: | |||
***Use 480 uL beads to DNA (1:1) | |||
***2 washes of 800 uL 80% EtOH | |||
***Elute in 100 uL LowTE | |||
* Sonication (2014-05-01) | |||
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power | |||
[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]] | |||
1) 1uL pre-sheared DNA, | |||
2) 2uL 100bp low scale ladder, | |||
3) 3uL sheared DNA | |||
*Blunt End Repair (2014-05-01) | |||
**Mix: | |||
***50.0 uL fragmented DNA | |||
***5.5 uL water | |||
***3.0 uL End Prep Enzyme Mix | |||
***6.5 uL End Repair Reaction Buffer | |||
**Incubation: | |||
***20 degrees for 30 minutes | |||
***65 degrees for 30 minutes | |||
*P2 adapter ligation (2014-05-01) | |||
**Add to mix: | |||
***15.0 uL Blunt/TA Ligase Master Mix | |||
***2.5 uL P2 RAD adapter (5 uM) | |||
***1.0 uL Ligation Enhancer | |||
**Incubation: | |||
***20 degrees for 15 minutes | |||
*Size selection (2014-05-01) | |||
**Add 16.5 uL water for a total of 100 uL | |||
**AMPure bead size selection: | |||
***45 uL beads to remove large fragments | |||
***25 uL beads to remove small fragments | |||
***3 washes of 200 uL 80% EtOH | |||
***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 | |||
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Revision as of 16:36, 1 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
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