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

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'''Program'''
'''Program'''
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold<br>
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
* 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

Revision as of 23:36, 5 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/Noi/NOTES/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


<--! ===Bead Purify + Measure Conc=== Following: Purification Protocol
4 tubes of 50ul with 50ul beads
Elute with 12.5ul H20 in each tube to make 50ul total
-->