Matt:LabNotes/2015-1-26: Difference between revisions
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'''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold | '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold | ||
[[File:012614_CA12kNov14_ExpansionPCR_V6V8.JPG]] | [[File:012614_CA12kNov14_ExpansionPCR_V6V8.JPG|650px]] | ||
*Did not think V6 amplified correctly (possibly due to underpipetting a primer) but [[Matt:LabNotes/2015-1-5#Expansion_PCR|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 | *Purified each with 1 Qiagen column and eluted with 50ul H2O | ||
*Nanodrop: | *Nanodrop: |
Revision as of 22:29, 26 January 2015
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