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| Line 6: |
Line 6: |
| | colspan="2"| | | | colspan="2"| |
| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| ==RAD Library Prep - Skinks Plate 1== | | ==Praomys E-Gel== |
| * 96 well plate of 50ng of DNA 10uL | | * Results of collecting DNA band from Praomys extractions using E-Gel SizeSelect: |
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| |
|
| * Digestion (2015-01-14)
| | [[Image:PraomysEgel04 10 2015.jpg|500px]] |
| **Mix and add to each well:
| |
| ***0.68 uL water
| |
| ***1.20 uL NEBuffer 4
| |
| ***0.12 uL SbfI-HF
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| **Incubation (RADIGEST on TONY):
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| ***37 degrees for 60 minutes
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| ***65 degrees for 20 minutes
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| |
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| * P1 adapter ligation (2015-01-14)
| |
| **Add 2 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
| |
| **Mix and add to each well:
| |
| ***1.28 uL water
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| ***0.40 uL NEBuffer 4
| |
| ***0.16 uL ATP
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| ***0.16 uL T4 Ligase
| |
| **Incubation (RADLIG on TONY):
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| *** 20 degrees for 60 minutes
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| *** 65 degrees for 20 minutes
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| |
|
| * Clean up (2015-01-15)
| | Samples labeled with the letter 'A' were collected from the E-Gel. |
| **Take 5 uL from each well and combine to 1.7 mL tube
| | The samples without an 'A' at the end of the name were the original extractions before running the E-Gel. |
| **AMPure bead clean up:
| |
| ***Used 440 uL beads to DNA (1:1)
| |
| ***2 washes of 800 uL 80% EtOH
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| ***Elute in 100 uL LowTE
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|
| |
|
| * Sonication (2015-01-15)
| | Top set of wells: |
| **BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
| |
| [[Image:Skinks sheared1 2015-01-15.JPG|150px]]
| |
| 1) 2uL 100bp low scale ladder,
| |
| 2) 2uL sheared DNA
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| <br>
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| 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
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|
| |
|
| *Blunt End Repair (2015-01-15)
| | 1) 2ul of CM4, 2) 2ul of CM4-A, 3) 2ul of CM5, 4) 2ul of CM5-A, 5) 2 uL 100 bp low scale ladder, 6) 2ul of GC11, 7) 2ul of GC11-A, 8) 2ul of GC13, 9) 2ul of GC13-A, 10) Empty |
| **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 (NEBENDRP on JOHN Block B):
| |
| ***20 degrees for 30 minutes
| |
| ***65 degrees for 30 minutes
| |
|
| |
|
| *P2 adapter ligation (2015-01-15)
| | Bottom set of wells: |
| **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 (NEBLIGAT on JOHN Block B):
| |
| ***20 degrees for 15 minutes
| |
| | |
| *Size selection (2015-01-15)
| |
| **Add 16.5 uL water for a total of 100 uL
| |
| **AMPure bead size selection:
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| ***45 uL beads to remove large fragments
| |
| ***25 uL beads to remove small fragments
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| ***3 washes of 200 uL 80% EtOH
| |
| ***Elute in 20 uL LowTE
| |
| | |
| *PCR amplification (2015-01-15)
| |
| **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 (NEBPCR 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
| |
| [[Image:Skinks pcr 2015-01-15.JPG|200px]]
| |
| 1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
| |
| | |
| *Bead clean up (2015-01-16)
| |
| **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. 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)
| |
|
| |
|
| | 1) 2ul of KS7, 2) 2ul of KS7-A, 3) 2ul of KS22, 4) 2ul of KS22-A, 5) 2 uL 100 bp low scale ladder, 6) 2ul of MC1, 7) 2ul of MC1-A, 8) 2ul of MC2, 9) 2ul of MC2-A, 10) Empty |
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| <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
PIRE RAD Library Preps
