Matt:LabNotes/2015-1-26
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CA12k_Nov2014 RevComp Probe Production
- Production of Padlock Probes targeting cDNA (V6 and V8 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:
- V6 primers - CA12k_Nov2014_Contig_RevComp
- V8 primers - CA12k_Nov2014_Const_RevComp
' | Contig of Exons RevComp(V6) | Constitutive Exons RevComp(V8) |
CA12k_Nov2014 Seed Oligos (900nM) | 15 | 15 |
2X KAPA SYBG MM | 67.5 | 67.5 |
100uM AP1V6U | 0.6 | 0 |
100uM AP2V6 | 0.6 | 0 |
100uM AP1V8U | 0 | 0.6 |
100uM AP2V8 | 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:012614 CA12kNov14 ExpansionPCR V6V8.JPG
- Did not think V6 amplified correctly (possibly due to underpipetting a primer) but V4 showed a similar curve
- Nanodrop also shows V6 amplified as much or more than V4
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM
- V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM
- Diluted to 10nM
- V6: 48ul x 95nM = 456ul x 10nM
- Add 408ul H2O
- V8: 48ul x 129nM = 619ul x 10nM
- Add 571ul H2O
- V6: 48ul x 95nM = 456ul x 10nM
Gel Check
- Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
- Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control
File:2015-01-26 1stRndAmplicon V6V8 Gel.jpg
- Apparently Nanodrop is not accurate enough at low conc
- V6 is missing a band at 188bp...
- Try Expansion PCR with V6 primers on the last of the seed oligos
V6 Expansion PCR Try 2
Components | Volume |
CA12k_Nov2014 Seed Oligos (900nM) | 14 |
2X KAPA SYBG MM | 50 |
100uM AP1V6U | 1 |
100uM AP2V6 | 1 |
H2O | 34 |
Total | 100 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold File:012614 CA12kNov14 ExpansionPCR V6 try2.JPG
- Stopped after 22 cycles
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V6: 17.9 ng/ul -> 17.9 ng/ul / (188bp*660Da/bp) = 144nM
- Diluted to 10nM
- V6: 48ul x 144nM = 691ul x 10nM
- Add 643ul H2O
- V6: 48ul x 144nM = 691ul x 10nM
Production PCR
- V6 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V6 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V6U (100uM) | 0.4 | 20 |
AP2V6 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- V8 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V8 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V8U (100uM) | 0.4 | 20 |
AP2V8 (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:012714 CA12kNov14 ProductionPCR V6V8.JPG
EtOH Precipitation
- 6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 1hr
- 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 V6 probes into one 1.5ml and all V8 probes into another
- Measured concentration with Nanodrop:
~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug
~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug
Lambda Exonuclease Digestion
- Divide each into 5 pcr tubes of 58ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 58 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 22 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 5 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- ng/ul x ul = ug (% yield)
- ng/ul x ul = ug (% yield)