Matt:LabNotes/2015-1-26: Difference between revisions
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'''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold | '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold | ||
[[File:012614_CA12kNov14_ExpansionPCR_V6_try2.JPG|650px]] | [[File:012614_CA12kNov14_ExpansionPCR_V6_try2.JPG|650px]] | ||
*Stopped after 22 cycles | |||
*Purified each with 1 Qiagen column and eluted with 50ul H2O | *Purified each with 1 Qiagen column and eluted with 50ul H2O | ||
*Nanodrop: | *Nanodrop: | ||
Line 81: | Line 82: | ||
**V6: 48ul x 144nM = '''691ul''' x 10nM | **V6: 48ul x 144nM = '''691ul''' x 10nM | ||
***Add 643ul H2O | ***Add 643ul H2O | ||
===Production PCR=== | ===Production PCR=== | ||
* | *V6 master mix | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 89: | Line 90: | ||
| align="center" style="background:#f0f0f0;"|'''Volume (50X)''' | | align="center" style="background:#f0f0f0;"|'''Volume (50X)''' | ||
|- | |- | ||
| First round amplicon | | First round amplicon CA12k_Nov14_V6 (10nM)||0.2||10 | ||
|- | |- | ||
| 2X KAPA SYBG MM||50||2500 | | 2X KAPA SYBG MM||50||2500 | ||
|- | |- | ||
| | | AP1V6U (100uM)||0.4||20 | ||
|- | |- | ||
| | | AP2V6 (100uM)||0.4||20 | ||
|- | |- | ||
| H2O||49||2450 | | H2O||49||2450 | ||
Line 101: | Line 102: | ||
| Total||100||5000 | | Total||100||5000 | ||
|} | |} | ||
* | *V8 master mix | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 107: | Line 108: | ||
| align="center" style="background:#f0f0f0;"|'''Volume (50X)''' | | align="center" style="background:#f0f0f0;"|'''Volume (50X)''' | ||
|- | |- | ||
| First round amplicon | | First round amplicon CA12k_Nov14_V8 (10nM)||0.2||10 | ||
|- | |- | ||
| 2X KAPA SYBG MM||50||2500 | | 2X KAPA SYBG MM||50||2500 | ||
|- | |- | ||
| | | AP1V8U (100uM)||0.4||20 | ||
|- | |- | ||
| | | AP2V8 (100uM)||0.4||20 | ||
|- | |- | ||
| H2O||49||2450 | | H2O||49||2450 | ||
Line 120: | Line 121: | ||
|} | |} | ||
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold | ||
*Stopped after 15 cycles | <!--*Stopped after 15 cycles | ||
[[File:010514_CA12kNov14_ProductionPCR_V4V7.JPG | 800px]] | [[File:010514_CA12kNov14_ProductionPCR_V4V7.JPG | 800px]] | ||
Revision as of 19:43, 27 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
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