CTR:Notebook/PIRE/Entry Base: Difference between revisions
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==RAD Library Prep - Skinks Plate | ==RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)== | ||
* | * 48 wells with 100ng of DNA in 20uL | ||
* Digestion (2015- | * 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 (RADIGEST on TONY): | **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 (2015- | * 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 (RADLIG on TONY): | **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 (2015- | * 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 | ***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 (2015- | * 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) 2uL 100bp low scale ladder, | 1) 2uL 100bp low scale ladder, | ||
2) 2uL sheared DNA | 2) 2uL sheared DNA | ||
<br> | <br> | ||
Additional | *Additional 2 cycles of 30 seconds on, 90 seconds off, High Power<br> | ||
[[Image: | [[Image:Sheared08262015 02.JPG|100px]] | ||
1) 2uL 100bp low scale ladder, | 1) 2uL 100bp low scale ladder, | ||
2) 2uL sheared DNA | 2) 2uL sheared DNA | ||
*Blunt End Repair (2015- | *Blunt End Repair (2015-08-26) | ||
**Mix: | **Mix: | ||
***50.0 uL fragmented DNA | ***50.0 uL fragmented DNA | ||
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***65 degrees for 30 minutes | ***65 degrees for 30 minutes | ||
*P2 adapter ligation (2015- | *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 | ||
Line 65: | Line 65: | ||
***20 degrees for 15 minutes | ***20 degrees for 15 minutes | ||
*Size selection (2015- | *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 (2015- | *PCR amplification (2015-08-26) | ||
**Mix: | **Mix: | ||
***5 uL DNA | ***5 uL DNA | ||
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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: | **Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel. | ||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product | [[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) | |||
*Re-run | |||
**Mix: | **Mix: | ||
***10 uL DNA | ***10 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) | ||
[[Image:PCRproduct08272015 2.JPG|200px]] | |||
[[Image: | 1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br> | ||
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product | →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 | *PicoGreen: 3.15 ng/μL | ||
Revision as of 09:19, 31 August 2015
PIRE RAD Library Preps | <html><img src="/images/9/94/Report.png" border="0" /></html> 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
|