CTR:Notebook/PIRE/2014/07/09: Difference between revisions

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==RAD Library Prep - Olive Sunbirds Plate 1==
==RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2==
* 96 well plate of 50ng of DNA 10uL
* 96 well plate of 50ng of DNA 10uL
 
<br>
* Digestion (2014-04-30)
<b>For Digestion & Ligation steps:
*Sunbirds Plate 2 - 2014-07-09, Sork thermocycler
*Greenbuls Plate 1 - 2014-07-11, Tony thermocycler
*Greenbuls Plate 2 - 2014-07-14, Sork thermocycler</b>
<br>
* Digestion
**Mix and add to each well:
**Mix and add to each well:
***0.68 uL water
***0.68 uL water
Line 18: Line 23:
***65 degrees for 20 minutes
***65 degrees for 20 minutes


* P1 adapter ligation (2014-04-30)
* P1 adapter ligation
**Add 2 uL indexed P1 adapter (10nM) to each well
**Add 2 uL indexed P1 adapter (10nM) to each well
**Mix and add to each well:
**Mix and add to each well:
Line 28: Line 33:
*** 20 degrees for 60 minutes
*** 20 degrees for 60 minutes
*** 65 degrees for 20 minutes
*** 65 degrees for 20 minutes
<br>
<b>The rest of the steps were done with all 3 libraries:</b>


* Clean up (2014-04-30)
* Clean up - 2014-07-15
**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:
***Use 480 uL beads to DNA (1:1)
***Use ~460 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 (2014-05-01)
* Sonication - 2014-07-15
**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:Sunbird1 sheared 2014-05-01.JPG|200px]]  
[[Image:RADbirds-shearinggels.png‎|500px]]  
1) 1uL pre-sheared DNA,
2μL of sheared DNA in each well
2) 2uL 100bp low scale ladder,
<br>
3) 3uL sheared DNA
2014-07-16:
 
<br>
*Blunt End Repair (2014-05-01)
*Blunt End Repair  
**Mix:
**Mix:
***50.0 uL fragmented DNA
***50.0 uL fragmented DNA
***5.5 uL water
***5.5 uL LowTE
***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: 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 (2014-05-01)
*P2 adapter ligation  
**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: on John Block B
***20 degrees for 15 minutes
***20 degrees for 15 minutes


*Size selection (2014-05-01)
*Size selection  
**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 76:
***Elute in 20 uL LowTE
***Elute in 20 uL LowTE


*PCR amplification (2014-05-01)
*PCR amplification  
**Mix:
**Mix:
***5 uL DNA
***5 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)
**PCR cycle:
**PCR cycle: on John Block B
***98 degrees for 30 seconds
***98 degrees for 30 seconds
***15 cycles of:
***15 cycles of:
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****72 degrees for 30 seconds
****72 degrees for 30 seconds
***72 degrees for 5 minutes
***72 degrees for 5 minutes
[[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
[[Image:RADbirds-PCRs.png|400px]]<br>
1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product
1) 1 μL Sunbird Plate 2 template, 2) 5 μL Sunbird Plate 2 PCR product<br>
3) 1 μL Greenbul Plate 1 template, 4) 5 μL Greenbul Plate 1 PCR product<br>
5) 1 μL Greenbul Plate 2 template, 6) 5 μL Greenbul Plate 2 PCR product
<br>
 
 
<b>Troubleshooting</b>
*Tried re-prep of leftover sheared DNA (Blunt End Repair → PCR), but no difference
<br>
 
*Used old P1 primer in amplification
[[Image:PCR-oldP1primer.JPG|300px]]<br>
→ Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying.
<br>
 


*Bead clean up (2014-05-02)
*Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments.
**Use 45 uL AMPure beads (1:1)
[[Image:Sheared-greenbuls2.JPG|300px]]
**2 washes of 200 uL 80% EtOH
1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA<br>
**Elute in 30 uL LowTE
*1st shearing step looks good - proceed to Blunt End Repair → PCR
[[Image:PCR-greenbuls2.JPG|300px]]
1) 1μL template, 2) 5μL PCR product<br>
→ Still no amplification. Sonication is not the issue.
<br>


*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)
*Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer
[[Image:Goodtemplate P2test.JPG|300px]]
1&2) Sunbirds Plate 1 template and PCR product, 3&4) Greenbuls Plate 2 old template and PCR product<br>
→ PCR with good template still works. P2 primer only does not work at all


==RAD Library Prep - ==
==RAD Library Prep - ==

Revision as of 16:18, 30 July 2014

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RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2

  • 96 well plate of 50ng of DNA 10uL


For Digestion & Ligation steps:

  • Sunbirds Plate 2 - 2014-07-09, Sork thermocycler
  • Greenbuls Plate 1 - 2014-07-11, Tony thermocycler
  • Greenbuls Plate 2 - 2014-07-14, Sork thermocycler


  • Digestion
    • 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
    • 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


The rest of the steps were done with all 3 libraries:

  • Clean up - 2014-07-15
    • Take 5 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Use ~460 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication - 2014-07-15
    • BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power

2μL of sheared DNA in each well
2014-07-16:

  • Blunt End Repair
    • Mix:
      • 50.0 uL fragmented DNA
      • 5.5 uL LowTE
      • 3.0 uL End Prep Enzyme Mix
      • 6.5 uL End Repair Reaction Buffer
    • Incubation: on John Block B
      • 20 degrees for 30 minutes
      • 65 degrees for 30 minutes
  • P2 adapter ligation
    • 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: on John Block B
      • 20 degrees for 15 minutes
  • Size selection
    • 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
    • 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: on John Block B
      • 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


1) 1 μL Sunbird Plate 2 template, 2) 5 μL Sunbird Plate 2 PCR product
3) 1 μL Greenbul Plate 1 template, 4) 5 μL Greenbul Plate 1 PCR product
5) 1 μL Greenbul Plate 2 template, 6) 5 μL Greenbul Plate 2 PCR product


Troubleshooting

  • Tried re-prep of leftover sheared DNA (Blunt End Repair → PCR), but no difference


  • Used old P1 primer in amplification


→ Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying.


  • Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments.

1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA

  • 1st shearing step looks good - proceed to Blunt End Repair → PCR

1) 1μL template, 2) 5μL PCR product
→ Still no amplification. Sonication is not the issue.


  • Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer

1&2) Sunbirds Plate 1 template and PCR product, 3&4) Greenbuls Plate 2 old template and PCR product
→ PCR with good template still works. P2 primer only does not work at all

RAD Library Prep -
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