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