Matt:LabNotes/2015-6-29: Difference between revisions
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>Mzcai (Created page with "==CA12k_Nov2014_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering *Train Justin ===Production PCR=== *...") |
>Mzcai mNo edit summary |
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**80ul 3M NaOAc pH 5.2-5.5 | **80ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for overnight | *Vortexed and put in -80C for overnight | ||
*Centrifuged at 3000rpm at 4C for 30 min | *Centrifuged at 3000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH | ||
Line 49: | Line 49: | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
~ | ~585ul of V4 probes: 77.0 ng/ul => ~45 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
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*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with 6 | *Purified with 6 QIA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **42.8ng/ul x 240ul = 10.3ug (46% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
Line 108: | Line 108: | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for overnight (~15hrs) | *Incubated at 37C for overnight (~15hrs) | ||
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===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 30ul each column (115ul total) | *Eluted 30ul each column (115ul total) |
Revision as of 21:40, 30 June 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Train Justin
Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10000 |
- 2 wells were spilled so left empty
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:20150629 CA12kNov14 ProductionPCR V4.JPG
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each) for V4
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~585ul of V4 probes: 77.0 ng/ul => ~45 ug
Lambda Exonuclease Digestion
- Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 96 |
10X Lambda Exo Buffer | 12 |
Lambda Exonuclease | 12 |
Total | 120 |
- Incubated at 37C for 1hr
- Purified with 6 QIA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 42.8ng/ul x 240ul = 10.3ug (46% yield)
Remove Amplification Adapters
USER
- Split into 4 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 59 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 8 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V4 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (~15hrs)