Matt:LabNotes/2015-5-11: Difference between revisions
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*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with 4 Zymo ssDNA/RNA columns | *Purified with 4 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **92.9 ng/ul x 160ul = 14.9 ug (67% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
Line 104: | Line 104: | ||
*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 20ul each column (80ul total) | *Eluted 20ul each column (80ul total) |
Revision as of 23:29, 12 May 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V4 (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 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:20150511 CA12kNov2014 V4 ProductionPCR.JPG
EtOH Precipitation
- 8 5-ml tubes (with 12 wells of PCR product each) for V4
- 1200ul PCR product
- 3000ul 100% EtOH
- 3ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 13000rpm at 4C for 20 min (in basement ultracentrifuge)
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C
- Discarded supernatant and air-dried for 10 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~400ul of V4 probes: 111.1 ng/ul => ~44.4 ug
Lambda Exonuclease Digestion
- Divide into 4 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 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 92.9 ng/ul x 160ul = 14.9 ug (67% 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)