Matt:LabNotes/2013-6-6: Difference between revisions

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* 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


2 reactions of Dan's V6 probes:
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10nM Agilent Expansion PCRd 0 gap Oligos||0.20
|-
| 100uM Forward ISB_AP1V6_AFv2||0.40
|-
| 100uM Reverse ISB_AP2V6_AR||0.40
|-
| 2x KAPA SYBG fast MM||50
|-
| H2O||49
|-
| Total||100
|}
2 reactions of Dan's V4 probes:
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10nM Agilent Expansion PCRd 20 gap Oligos||0.20
|-
| 100uM Forward ISB_AP1V4_AFv2||0.40
|-
| 100uM Reverse ISB_AP2V4_AR||0.40
|-
| 2x KAPA SYBG fast MM||50
|-
| H2O||49
|-
| Total||100
|}
'''Dan's Probes Program'''
98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x8 -> 72C 3min
*13 cycles total
 
  *First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O


<!-- ===Bead Purify + Measure Conc===
<!-- ===Bead Purify + Measure Conc===
Following: [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/Bead_Purification_Protocol|Bead Purification Protocol] <br>
Following: [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/Bead_Purification_Protocol|Bead Purification Protocol] <br>
4 tubes of 50ul with 50ul beads <br>
16 tubes of 50ul with 50ul beads (2 tubes for each PCR)<br>
Elute with 12.5ul H20 in each tube to make 50ul total <br> -->
Elute with 12.5ul H20 in each tube to make 50ul total for each sample<br> -->

Revision as of 17:18, 6 June 2013

Sequence Agilent Oligos

PCR

PCR Mix

2 reactions of 0 gap probes:

Components Volume
10nM Agilent Expansion PCRd 0 gap Oligos 0.20
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 reactions of 20 gap probes:

Components Volume
10nM Agilent Expansion PCRd 20 gap Oligos 0.20
100uM Forward ISB_AP1V4_AFv1 0.40
100uM Reverse ISB_AP2V4_AR 0.40
2x KAPA SYBG fast MM 50
H2O 49
Total 100

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

  • 2 tubes of each in case concentration is low
  • Did same number of PCR cycles as Production PCR to keep amplification as consistent as possible


2 reactions of Dan's V6 probes:

Components Volume
10nM Agilent Expansion PCRd 0 gap Oligos 0.20
100uM Forward ISB_AP1V6_AFv2 0.40
100uM Reverse ISB_AP2V6_AR 0.40
2x KAPA SYBG fast MM 50
H2O 49
Total 100

2 reactions of Dan's V4 probes:

Components Volume
10nM Agilent Expansion PCRd 20 gap Oligos 0.20
100uM Forward ISB_AP1V4_AFv2 0.40
100uM Reverse ISB_AP2V4_AR 0.40
2x KAPA SYBG fast MM 50
H2O 49
Total 100

Dan's Probes Program 98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x8 -> 72C 3min

  • 13 cycles total
 *First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O