CTR:Notebook/PIRE/Entry Base: Difference between revisions
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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, using new plate of adapters made in-house | ||
**Mix and add to each well: | **Mix and add to each well: | ||
***1.28 uL water | ***1.28 uL water | ||
| Line 25: | Line 25: | ||
***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 (2015-01-15) | ||
**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: | ||
*** | ***Used 440 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 (2015-01-15) | ||
**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:Skinks sheared1 2015-01-15.JPG|150px]] | ||
1) | 1) 2uL 100bp low scale ladder, | ||
2) 2uL sheared DNA | |||
<br> | |||
Additional 3 cycles of 15 seconds on, 90 seconds off, High Power<br> | |||
[[Image:Skinks sheared2 2015-01-15.JPG|150px]] | |||
1) 2uL 100bp low scale ladder, | |||
2) 2uL sheared DNA | |||
*Blunt End Repair ( | *Blunt End Repair (2015-01-15) | ||
**Mix: | **Mix: | ||
***50.0 uL fragmented DNA | ***50.0 uL fragmented DNA | ||
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***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 (NEBENDRP 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 (2015-01-15) | ||
**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 (NEBLIGAT on JOHN Block B): | ||
***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection ( | *Size selection (2015-01-15) | ||
**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 73: | ||
***Elute in 20 uL LowTE | ***Elute in 20 uL LowTE | ||
*PCR amplification ( | *PCR amplification (2015-01-15) | ||
**Mix: | **Mix: | ||
***5 uL DNA | ***5 uL DNA | ||
| Line 76: | Line 80: | ||
*** 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 (NEBPCR on JOHN Block B): | ||
***98 degrees for 30 seconds | ***98 degrees for 30 seconds | ||
***15 cycles of: | ***15 cycles of: | ||
| Line 83: | Line 87: | ||
****72 degrees for 30 seconds | ****72 degrees for 30 seconds | ||
***72 degrees for 5 minutes | ***72 degrees for 5 minutes | ||
[[Image: | [[Image:Skinks pcr 2015-01-15.JPG|200px]] | ||
1 | 1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product | ||
*Bead clean up ( | *Bead clean up (2015-01-16) | ||
**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 | ||
**Elute in 30 uL LowTE | **Elute in 30 uL LowTE | ||
*Quant DNA library using PicoGreen | *Quant DNA library using PicoGreen: 1.53 ng/μL. Using Qubit HS: 2.5 ng/μL. <b>→ Too low to get 10nm in 10μL.</b> | ||
<br> | |||
*Re-run of PCR using 10μL template DNA (2015-01-16) | |||
**Mix: | |||
***10 uL DNA | |||
***25 uL NEBNext High Fidelity 2x PCR Master Mix | |||
***13 uL water | |||
*** 1 uL P1 adapter primer (25 uM) | |||
*** 1 uL P2 adapter primer (25 uM) | |||
**PCR cycle: NEBPCR on JOHN Block B | |||
[[Image:Skinks pcr 2015-01-16.JPG|200px]] | |||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product | |||
*PicoGreen: 3.15 ng/μL | |||
<br><br> | |||
*Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06) | |||
[[Image:Bioanalyzer-skinks-plate1.jpg|450px]] | |||
*Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2015-02-09) | |||
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Revision as of 15:06, 1 April 2015
 PIRE RAD Library Preps
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RAD Library Prep - Skinks Plate 1
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