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>


2 reactions of 0 gap probes:
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||0.20
| 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
|}
|}


2 reactions of 20 gap probes:
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||0.20
| 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
* 2 tubes of each in case concentration is low
* 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


<!-- ===Bead Purify + Measure Conc===
==20 gap Probe Capture Reaction==
Following: [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/Bead_Purification_Protocol|Bead Purification Protocol] <br>
Followed plan from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4
4 tubes of 50ul with 50ul beads <br>
Elute with 12.5ul H20 in each tube to make 50ul total <br> -->

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

20 gap Probe Capture Reaction[edit]

Followed plan from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4