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)