Matt:LabNotes/2015-1-5
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CA12k_Nov2014 Probe Production
- Production of Padlock Probes targeting cDNA (V4 and V7 primers) of Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Expansion PCR
- Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
- Make 2 tubes:
- V4 primers - CA12k_Nov2014_Contig
- V7 primers - CA12k_Nov2014_Const
' | Contig of Exons (V4) | Constitutive Exons (V7) |
CA12k_Nov2014 Seed Oligos (900nM) | 15 | 15 |
2X KAPA SYBG MM | 67.5 | 67.5 |
100uM AP1V4U | 0.6 | 0 |
100uM AP2V4 | 0.6 | 0 |
100uM AP1V7U | 0 | 0.6 |
100uM AP2V7 | 0 | 0.6 |
H2O | 51.3 | 51.3 |
Total | 135 | 135 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold
File:010514 CA12kNov14 ExpansionPCR V4V7.JPG
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V4: 10.8 ng/ul -> 10.8 ng/ul / (188bp*660Da/bp) = 87nM
- V7: 17.8 ng/ul -> 17.8 ng/ul / (190bp*660Da/bp) = 142nM
- Diluted to 10nM
- V4: 50ul x 87nM = 435ul x 10nM
- Add 385ul H2O
- V7: 50ul x 142nM = 710ul x 10nM
- Add 660ul H2O
- V4: 50ul x 87nM = 435ul x 10nM
Production PCR
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- 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 22 -> 72C 2min -> 15C hold
- Stopped after 15 cycles
File:010514 CA12kNov14 ProductionPCR V4V7.JPG
EtOH Precipitation
- 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes 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 12 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 V4 probes into one 1.5ml and all V7 probes into another
- Measured concentration with Nanodrop:
~290ul of V4 probes: 127.8 ng/ul => ~37 ug ~290ul of V7 probes: 113.9 ng/ul => ~33 ug
Lambda Exonuclease Digestion
- Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 72.5 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 7.5 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- ng/ul x ul = ug (63.6% yield)