Dinh/NOTES/2010-1-4

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Purpose[edit]

Due to recent low capturing efficiency of CpG30K set, we want to further optimize the capturing protocol for this subset to obtain better capturing efficiency. Also, for future pipelining of the capturing protocol, the optimal template and probes concentration to work with should be identified so that future capturing experiments can use similar concentration ranges and obtain less variability in results.

Capturing Optimization for CpG30K set[edit]

CpG30K probes set_12/30, 30ng/ul 
200ng templates, 200ng/ul bisulfite converted Jurkat
100 templates to probes ratio
Capturing System 1x 9x
Volume of probes 0.37 3.3
Volume of templates 1 9
Volume of suppressor oligo 1 9
Volume of H20 6.63 59.7
Volume of 10X Ampligase buffer 1 9
Total Volume 10 90

Experimental Setup[edit]

1. Test different dNTP concentration
2. Test whether different cycle # affect non-specific capturing
Conditions 1 2 NTC
A 0.5mM dNTP in SLN Mix, no cycling 0.5mM dNTP in SLN Mix, 6 cycles 0.5mM dNTP (-) No cycling
B 0.2mM dNTP in SLN Mix, no cycling 0.2mM dNTP in SLN Mix, 6 cycles 0.2mM dNTP (-) 6 cycles
C 0.2mM dNTP in 1st SLN Mix, 0.5mM in 2nd, 4 cycles 0.2mM dNTP in SLN Mix, 4 cycles 0.2mM dNTP (-) 4 cycles

SLN Mix

' volume to add for 1ul of 0.5mM dNTP SLN volume to add for 1ul of 0.2mM dNTP SLN
dH2O 0.1 0.4
dNTP (1mM) 0.5 0.2
10x AmpliLigase Buffer 0.1 0.1
AmpLigase (5U/ul) 0.1 0.1
Stoffel (10U/ul) 0.2 0.2

Program

1. 95C->10min
2. 55C->forever (20hrs), add 2 ul SLN mix
3. 55C->4 hr, add 2 ul of SLN mix (if cycling)
4. 95C->1min
5. 55C->4hr
6. Goto 4, 5x
7. 55C->forever (proceed to next step once 2ul of EXO I/III mix is ready)
8. 95C->5min
10. 37C->1hr, as soon as temp get to 37C add 2ul EXO I/III mix
11. 94C->5min
12. 4C forever