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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 Library Preps</span> | ||
|style="background-color: #F2F2F2" align="center"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==RAD Library Prep - | ==RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)== | ||
* | * 48 wells with 100ng of DNA in 20uL | ||
* Digestion ( | * Digestion (2015-08-25) | ||
**Mix and add to each well: | **Mix and add to each well: | ||
*** | ***1.36 uL water | ||
*** | ***2.40 uL CutSmart Buffer | ||
***0. | ***0.24 uL SbfI-HF (new) | ||
**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 (2015-08-25) | ||
**Add | **Add 4 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: | ||
*** | ***2.56 uL water | ||
***0. | ***0.8 uL NEBuffer 4 | ||
***0. | ***0.32 uL ATP | ||
***0. | ***0.32 uL T4 Ligase (new) | ||
**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 (2015-08-26) | ||
**Take | **Take 10 uL from each well and combine to 1.7 mL tube | ||
**AMPure bead clean up: | **AMPure bead clean up: | ||
*** | ***Used 430 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 - edited to get smaller average fragments (2015-08-26) | ||
**BioRuptor NGS: | **BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power | ||
[[Image: | [[Image:Sheared08262015 01.JPG|150px]] | ||
1) | 1) 2uL 100bp low scale ladder, | ||
2) 2uL sheared DNA | |||
<br> | |||
*Additional 2 cycles of 30 seconds on, 90 seconds off, High Power<br> | |||
[[Image:Sheared08262015 02.JPG|100px]] | |||
1) 2uL 100bp low scale ladder, | |||
2) 2uL sheared DNA | |||
*Blunt End Repair ( | *Blunt End Repair (2015-08-26) | ||
**Mix: | **Mix: | ||
***50.0 uL fragmented DNA | ***50.0 uL fragmented DNA | ||
Line 49: | Line 53: | ||
***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 (2015-08-26) | ||
**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 - edited to select for smaller insert (2015-08-26) | ||
**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: | ||
*** | ***55 uL beads to remove large fragments | ||
***25 uL beads to remove small fragments | ***25 uL beads to remove small fragments | ||
***3 washes of 200 uL 80% EtOH | ***3 washes of 200 uL 80% EtOH | ||
***Elute in 20 uL LowTE | ***Elute in 20 uL LowTE | ||
*PCR amplification ( | *PCR amplification (2015-08-26) | ||
**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 | ||
**Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel. | |||
[[Image:PCRproduct08272015.JPG|200px]] | |||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br> | |||
→Visible amplification, but size range difficult to discern. | |||
*Re-run PCR to improve visibility and get more amplification (2015-08-27) | |||
**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) | |||
[[Image:PCRproduct08272015 2.JPG|200px]] | |||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br> | |||
→Successful amplification. Clean up, quant, and Bioanalyzer will be done with other libraries. | |||
*Bead clean up | |||
**Use 45 uL AMPure beads (1:1) | |||
**2 washes of 200 uL 80% EtOH | |||
**Elute in 30 uL LowTE | |||
<br> | |||
*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) | |||
Latest revision as of 23:56, 26 September 2017
PIRE RAD Library Preps | Main project page |
RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product →Visible amplification, but size range difficult to discern.
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
|