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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Library Preps</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Adapter Preparation </span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==RAD Library Prep - Olive Sunbirds Plate 1==
==RAD P1 Adapter Ordering==
* 96 well plate of 50ng of DNA 10uL


* Digestion (2014-04-30)
[http://openwetware.org/images/e/eb/Multiple_Plate-20141120_RAD.xls Adapter Sequences] (sequences from Mike Miller's files)<br>
**Mix and add to each well:
Ordered in plate format from IDT (1 plate of top oligos, 1 plate of bottom oligos)<br>
***0.68 uL water
Parameters:
***1.20 uL NEBuffer 4
*Scale: 100 nmole
***0.12 uL SbfI-HF
*Purification: Standard desalting
**Incubation:
*Ship option: Wet
***37 degrees for 60 minutes
*Amount per well: Full yield
***65 degrees for 20 minutes
*Concentration: 100 μM
*Dilutant: IDTE Buffer pH 8.0


* P1 adapter ligation (2014-04-30)
==RAD P1 Adapter Annealing and Dilution==
**Add 2 uL indexed P1 adapter (10nM) to each well
Based on instructions from Mike Miller's RAD adapter information file.<br>
**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)
1. Dilute oligo plates from 100μM to 10μM
**Take 5 uL from each well and combine to 1.7 mL tube
*To make 20μL plate, take 2μL of 100μM stock and dilute in 18μL dH<sub>2</sub>O.
**AMPure bead clean up:
2.Make adapter mix plate (20μL)
***Use 480 uL beads to DNA (1:1)
*For each well:
***2 washes of 800 uL 80% EtOH
**To get 10mM Tris, need 0.2μL of 1M stock Tris
***Elute in 100 uL LowTE
**To get 50mM NaCl, need 0.2μL of 5M stock NaCl
 
**To get 2μM each oligo, need 4μL of 10μM each oligo
* Sonication (2014-05-01)
*For 96 well plate, make a 110x mix of 22μL of 1M Tris, 22μL of 5M NaCl, and 1276μL of dH<sub>2</sub>O into a microtube, then transfer to reservoir. Pipette 12μL of mix into each well, then add 4μL of top and bottom oligo to each well for a total of 20μL.
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
3. Anneal adapters
[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]
*98°C for 2 minutes
1) 1uL pre-sheared DNA,
*Ramp: 98°C to 10°C for 20 minutes
2) 2uL 100bp low scale ladder,
4. Make dilution plate (195μL of 10mM Tris, 50mM NaCl)
3) 3uL sheared DNA
*For each well, need:
 
**1.95μL of 1M Tris
*Blunt End Repair (2014-05-01)
**1.95μL of 5M NaCl
**Mix:
**191.1μL of dH<sub>2</sub>O
***50.0 uL fragmented DNA
*For 96 well plate, make 110x mix of 214.5μL of 1M Tris, 214.5μL of 5M NaCl, and 21.021mL dH<sub>2</sub>O into falcon tube, then transfer to reservoir. Pipette 195μL of mix into each well.
***5.5 uL water
*Add 5μL of annealed adapter to the dilution plate. The adapter concentration is now 50nM.
***3.0 uL End Prep Enzyme Mix
5. Make final adapter plates (10nM)
***6.5 uL End Repair Reaction Buffer
*For each plate, take 2μL of 50nM stock adapter and dilute in 8μL of dH<sub>2</sub>O.  
**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
 
*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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Latest revision as of 00:32, 27 September 2017

PIRE RAD Adapter Preparation Main project page
Previous entry      Next entry

RAD P1 Adapter Ordering

Adapter Sequences (sequences from Mike Miller's files)
Ordered in plate format from IDT (1 plate of top oligos, 1 plate of bottom oligos)
Parameters:

  • Scale: 100 nmole
  • Purification: Standard desalting
  • Ship option: Wet
  • Amount per well: Full yield
  • Concentration: 100 μM
  • Dilutant: IDTE Buffer pH 8.0

RAD P1 Adapter Annealing and Dilution

Based on instructions from Mike Miller's RAD adapter information file.

1. Dilute oligo plates from 100μM to 10μM

  • To make 20μL plate, take 2μL of 100μM stock and dilute in 18μL dH2O.

2.Make adapter mix plate (20μL)

  • For each well:
    • To get 10mM Tris, need 0.2μL of 1M stock Tris
    • To get 50mM NaCl, need 0.2μL of 5M stock NaCl
    • To get 2μM each oligo, need 4μL of 10μM each oligo
  • For 96 well plate, make a 110x mix of 22μL of 1M Tris, 22μL of 5M NaCl, and 1276μL of dH2O into a microtube, then transfer to reservoir. Pipette 12μL of mix into each well, then add 4μL of top and bottom oligo to each well for a total of 20μL.

3. Anneal adapters

  • 98°C for 2 minutes
  • Ramp: 98°C to 10°C for 20 minutes

4. Make dilution plate (195μL of 10mM Tris, 50mM NaCl)

  • For each well, need:
    • 1.95μL of 1M Tris
    • 1.95μL of 5M NaCl
    • 191.1μL of dH2O
  • For 96 well plate, make 110x mix of 214.5μL of 1M Tris, 214.5μL of 5M NaCl, and 21.021mL dH2O into falcon tube, then transfer to reservoir. Pipette 195μL of mix into each well.
  • Add 5μL of annealed adapter to the dilution plate. The adapter concentration is now 50nM.

5. Make final adapter plates (10nM)

  • For each plate, take 2μL of 50nM stock adapter and dilute in 8μL of dH2O.
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