Dinh/NOTES/2010-1-11
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Purpose[edit]
Characterizing CpG97K probes sets A and B's capturing efficiency at 55C vs 60C.
Capturing Optimization for CpG90K A/B sets[edit]
CpG97K A probes set (47ng/ul, Ida) CpG97K B probes set (62ng/ul, Ida) 300-400ng input templates 200 templates to probes ratio
Bis16 (309ng/ul) | Jurkat (280ng/ul) | |
Template input (ng) | 370.8 | 448 |
CpG97K A | CpG97K B | |
Probes input (ng) | 61.1 | 49.6 |
Reaction setup
10x AmpLigase buffer | Template | CpG97K A | CpG97K B | supp oligo A | supp oligo B | H2O | TOTAL | |
Bis16A (qPCR, 309ng/ul) | 2.5 | 3 | 3.25 | 0 | 2.5 | 0 | 13.75 | 25 |
Bis16B (qPCR, 309ng/ul) | 2.5 | 3 | 0 | 2 | 0 | 2.5 | 15 | 25 |
NTC_A | 2.5 | 0 | 3.25 | 0 | 2.5 | 0 | 16.75 | 25 |
NTC_B | 2.5 | 0 | 0 | 2 | 0 | 2.5 | 18 | 25 |
Jurkat_A (280ng/ul) | 2.5 | 4 | 3.25 | 0 | 2.5 | 0 | 12.75 | 25 |
Jurkat_B (280ng/ul) | 2.5 | 4 | 0 | 2 | 0 | 2.5 | 14 | 25 |
Strip 1 (55C)
Bis16A Bis16B NTC_A NTC_B J_A J_B blank blank
Strip 2 (60C)
Bis16A Bis16B NTC_A NTC_B J_A J_B blank blank
SLN Mix | 1x | 25x | Final Conc |
H2O | 0.5 | 12.5 | |
dNTP(10mM) | 0.1 | 2.5 | 1mM |
10xBuffer | 0.1 | 2.5 | 1x |
Ligase | 0.1 | 2.5 | 0.5U/ul |
Stoffel | 0.2 | 5 | 2U/ul |
TOTAL | 1 | 25 |
Program
1. 95C->10min 2. 55C/60C->forever (40hrs, starts 7pm (1/10) and ends at 11am (1/12)), add 2 ul SLN mix 3. 55C/60C->4 hr 4. 55C/60C->forever (proceed to next step once 2ul of EXO I/III mix is ready) 5. 95C->5min 6. 37C->1hr, as soon as temp get to 37C add 2ul EXO I/III mix 7. 94C->5min 8. 4C forever
PCR Amplification
Reagent | Vol (1x) | Vol (14x) |
Template (*) | 10 | |
2x iProof MM | 50 | 700 |
AmpF6.3Sol(10uM) | 4 | 56 |
AmpR6.3Sol(10uM) | 4 | 56 |
50x SYBR Green | 0.8 | 11.2 |
Rnase Free H2O | 31.2 | 436.8 |
TOTAL | 100 | 1260 |
Results[edit]
Jan 12, 2010 File:CpG97K A - bis 16 vs bis Jurkat, 55 v 60.JPG File:CpG97K B - bis 16 vs bis Jurkat, 55 v 60.JPG
Product captured at 60C have 3-4 cycles different for both CpG97K sets A and B. Sets A and B have about 3 cycles different.
Jan 13, 2010 File:Dinh 2010-01-13 Capture Amplicon 97K 55C incubation.jpg File:Dinh 2010-01-13 Capture Amplicon 97K 60C incubation.jpg