Matt:LabNotes/2015-1-13: Difference between revisions
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>Mzcai m (→USER) |
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*Incubate at 37C for | *Incubate at 37C for 2.5 hours | ||
====DpnII==== | ====DpnII==== | ||
*Added 15ul of the following | *Added 15ul of the following |
Revision as of 04:28, 14 January 2015
CA12k_Nov2014_V7 Probe Production
- Production of Padlock Probes targeting constitutive exon cDNA (V7 primers) of Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 10nM 1st round amplicons
- V7 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V7U (100uM) | 0.4 | 20 |
AP2V7 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:011214 CA12kNov14 ProductionPCR V7.JPG
EtOH Precipitation
- 6 15-ml tubes (with 8 wells of PCR product each) for V7
- 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 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~290ul of V7 probes: 93.2 ng/ul => ~27 ug
Lambda Exonuclease Digestion
- Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 100 |
10X Lambda Exo Buffer | 12.5 |
Lambda Exonuclease | 12.5 |
Total | 125 |
- Incubated at 37C for 1hr
- Purified with 3 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 62.9ng/ul x 120ul = 7.5ug (56% yield)
Remove Amplification Adapters
USER
- Save 2ul for gel check later
- Split into 2 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_V7 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight