Matt:LabNotes/2013-6-6: Difference between revisions
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==Sequence Agilent Oligos== | ==Sequence Agilent Oligos== | ||
*Diluted Expansion PCR'd 0 and 20 gap oligos from 112nM and 123 nM, respectively, to 50ul of 10nM each in a separate tube | |||
===PCR=== | ===PCR=== | ||
'''PCR Mix'''<br> | '''PCR Mix'''<br> | ||
Reaction of 0 gap probes: | |||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| 10nM Agilent Expansion PCRd 0 gap Oligos||0. | | 10nM Agilent Expansion PCRd 0 gap Oligos||0.40 | ||
|- | |- | ||
| 100uM Forward ISB_AP1V6_AFv1||0.40 | | 100uM Forward ISB_AP1V6_AFv1||0.40 | ||
Line 19: | Line 21: | ||
| H2O||49 | | H2O||49 | ||
|- | |- | ||
| Total||100 | | Total||100.2 | ||
|} | |} | ||
Reaction of 20 gap probes: | |||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| 10nM Agilent Expansion PCRd 20 gap Oligos||0. | | 10nM Agilent Expansion PCRd 20 gap Oligos||0.40 | ||
|- | |- | ||
| 100uM Forward ISB_AP1V4_AFv1||0.40 | | 100uM Forward ISB_AP1V4_AFv1||0.40 | ||
Line 37: | Line 39: | ||
| H2O||49 | | H2O||49 | ||
|- | |- | ||
| Total||100 | | Total||100.2 | ||
|} | |} | ||
Line 44: | Line 46: | ||
'''Program''' | '''Program''' | ||
98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x10 -> 72C 3min | 98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x10 -> 72C 3min | ||
* Did same number of PCR cycles as [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11 Production PCR] to keep amplification as consistent as possible | * Did same number of PCR cycles as [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11 Production PCR] to keep amplification as consistent as possible | ||
===Dan's Probes=== | |||
*Set up reaction for his probes the same as for 0 gap and 20 gap Agilent probes with same volumes of probe, primer, H2O and KAPA MM | |||
*Took out Dan's samples after 13 cycles | |||
*Because Dan's had more oligos than I expected (4 sets, each set with V4 and V6 within them), I had to repeat sequencing barcodes and so assigned them as follows: | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Tube #''' | |||
| align="center" style="background:#f0f0f0;"|'''Probes (0.4ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Forward Primer 100uM (0.4ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Reverse Primer 100uM (0.4ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total Cycles''' | |||
|- | |||
| 1||0gap 10nM||ISB_AP1V6_AFv1||ISB_AP2V6_AR||15 | |||
|- | |||
| 2||20gap 10nM||ISB_AP1V4_AFv1||ISB_AP2V4_AR||15 | |||
|- | |||
| 3||#1-V6 20nM||ISB_AP1V6_AFv2||ISB_AP2V6_AR||13 | |||
|- | |||
| 4||#1-V4 20nM||ISB_AP1V4_AFv2||ISB_AP2V4_AR||13 | |||
|- | |||
| 5||#2-V6 20nM||ISB_AP1V6_AFv1||ISB_AP2V6_AR||13 | |||
|- | |||
| 6||#2-V4 20nM||ISB_AP1V4_AFv1||ISB_AP2V4_AR||13 | |||
|- | |||
| 7||#3-V6 20nM||ISB_AP1V6_AFv2||ISB_AP2V6_AR||13 | |||
|- | |||
| 8||#3-V4 20nM||ISB_AP1V4_AFv2||ISB_AP2V4_AR||13 | |||
|- | |||
| 9||#4-V6 20nM||ISB_AP1V6_AFv1||ISB_AP2V6_AR||13 | |||
|- | |||
| 10||#4-V4 20nM||ISB_AP1V4_AFv1||ISB_AP2V4_AR||13 | |||
|- | |||
| | |||
|} | |||
*First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O | |||
[[File:060613_AgiOligoSeq_qPCR.JPG | 450px]] | |||
[[File:060613_DansOligoSeq_qPCR.JPG | 650 px]] | |||
*Bead purification and gel verification tomorrow: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-7 | |||
==20 gap Probe Capture Reaction== | |||
Followed plan from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4 | |||
Latest revision as of 18:26, 7 June 2013
Sequence Agilent Oligos[edit]
- Diluted Expansion PCR'd 0 and 20 gap oligos from 112nM and 123 nM, respectively, to 50ul of 10nM each in a separate tube
PCR[edit]
PCR Mix
Reaction of 0 gap probes:
Components | Volume |
10nM Agilent Expansion PCRd 0 gap Oligos | 0.40 |
100uM Forward ISB_AP1V6_AFv1 | 0.40 |
100uM Reverse ISB_AP2V6_AR | 0.40 |
2x KAPA SYBG fast MM | 50 |
H2O | 49 |
Total | 100.2 |
Reaction of 20 gap probes:
Components | Volume |
10nM Agilent Expansion PCRd 20 gap Oligos | 0.40 |
100uM Forward ISB_AP1V4_AFv1 | 0.40 |
100uM Reverse ISB_AP2V4_AR | 0.40 |
2x KAPA SYBG fast MM | 50 |
H2O | 49 |
Total | 100.2 |
Agilent Oligos: Expansion PCRd but before Production PCR (see: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-10)
Program 98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x10 -> 72C 3min
- Did same number of PCR cycles as Production PCR to keep amplification as consistent as possible
Dan's Probes[edit]
- Set up reaction for his probes the same as for 0 gap and 20 gap Agilent probes with same volumes of probe, primer, H2O and KAPA MM
- Took out Dan's samples after 13 cycles
- Because Dan's had more oligos than I expected (4 sets, each set with V4 and V6 within them), I had to repeat sequencing barcodes and so assigned them as follows:
Tube # | Probes (0.4ul) | Forward Primer 100uM (0.4ul) | Reverse Primer 100uM (0.4ul) | Total Cycles |
1 | 0gap 10nM | ISB_AP1V6_AFv1 | ISB_AP2V6_AR | 15 |
2 | 20gap 10nM | ISB_AP1V4_AFv1 | ISB_AP2V4_AR | 15 |
3 | #1-V6 20nM | ISB_AP1V6_AFv2 | ISB_AP2V6_AR | 13 |
4 | #1-V4 20nM | ISB_AP1V4_AFv2 | ISB_AP2V4_AR | 13 |
5 | #2-V6 20nM | ISB_AP1V6_AFv1 | ISB_AP2V6_AR | 13 |
6 | #2-V4 20nM | ISB_AP1V4_AFv1 | ISB_AP2V4_AR | 13 |
7 | #3-V6 20nM | ISB_AP1V6_AFv2 | ISB_AP2V6_AR | 13 |
8 | #3-V4 20nM | ISB_AP1V4_AFv2 | ISB_AP2V4_AR | 13 |
9 | #4-V6 20nM | ISB_AP1V6_AFv1 | ISB_AP2V6_AR | 13 |
10 | #4-V4 20nM | ISB_AP1V4_AFv1 | ISB_AP2V4_AR | 13 |
*First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O
File:060613 AgiOligoSeq qPCR.JPG File:060613 DansOligoSeq qPCR.JPG
- Bead purification and gel verification tomorrow: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-7
20 gap Probe Capture Reaction[edit]
Followed plan from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4