Dinh/NOTES/2010-7-7

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Multiplexing Test 1[edit]

  • Tested AmpR6.3Ind1 and AmpR6.3Ind2 for clonal sequencing
  • Template: I used PGP1-iPS capture amplicons from CpG97K B probe set which was amplified once with AmpF/R6.3NH2

Reaction Mix[edit]

 0.5 ul template
 0.4 ul SYBR Green I
 2.0 ul AmpF6.3Sol
 2.0 ul AmpR6.3Ind1 or AmpR6.3Ind2
15.1 ul H2O
20.0 ul Phusion HF 2x MM
------------------------------------------
40.0 ul TOTAL

Program[edit]

98C - 30s
98C - 10s
58C - 20s
72C - 20s
go to 2 for 4x
98C - 10s
72C - 20s
go to 6 for 3x
72C - 3 min
15C - forever
----------------
(9 cycles total)

Results[edit]

File:Multiplexing 1 and 2.PNG
File:ZhangLab 2 2010-07-08 20hr 51min.jpg
File:ZhangLab 2 2010-07-08 20hr 58min.jpg
File:ZhangLab 2 2010-07-09 13hr 10min.jpg
2.15 ng/ul   PGP1-iPSInd1
2.00 ng/ul   PGP1-iPSInd2
4.44 ng/ul   PGP1-iPSInd1-reamp
4.91 ng/ul   PGP1-iPSInd2-reamp

Discussion[edit]

  • 58C might be too low (need to increase if amplifying from AmpF/R6.3NH2 products)
  • Over amplified products (increase first PCR annealing temperature and 4 additional cycles might be enough)
  • Need to do column purification before size selection
  • After 6% PAGE size selection, I reamplified the products for 4 more cycles of 98C (10s) -> 72C (20s)