Noi/NOTES/2014-3-19
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Experimental plan to test sensitivity of BSPP capture[edit]
- In the future, we want to apply BSPP to capture circulating DNA which are fragmented DNA and total amount for capture is very low. Therefore Dr. Zhang wanted to test the sensitivity of BSPP capture at different bisulfite converted DNA amount. At the same time, he wanted to improve the sensitivity of BSPP capture by performing low magnitude amplification with N9 primer and another option is doing multiple rounds of heat denaturation at 98C and circularization at 55C (I'll look into more details in previous published papers.).
- The sensitivity could be varied depends on the size of probe set and efficiency of BSPP as well. In this experiment, he wanted to use MONOD GP1_V4 probe set that is not too big (7,110 probes) and had been tested in previous experiment.
- I will be addressing the experiment to test HOTSPOTS 460k probe set later since I need to use this probe set to capture BSC samples from Illumina if the capture by this probe set work well. Otherwise, I will be using MONOD GP1_V4 to capture BSC samples.
- Since we’re pretty sure that 100-300ng work fine for capture, I am doing additional experiment to improve sensitivity of BSPP capture for the low input (<100ng).
- Summary Table:
- I will add more details of this experimental plan and ask Dr. Zhang's opinion if he agrees with this plan
Capture reaction set up[edit]
1) BSPP capture, STD protocol[edit]
- - Mix bis-cvt DNA with MONOD GP1_V4 in 1X Ampligase buffer (total volume 20ul)
- - Continue to BSPP capture, i) STD protocol or ii) multiple rounds of circularization
2) BSPP capture, low-magnitude amplification[edit]
- - Mix bis-cvt DNA with 200uM dNTP, 2mM N9, 2.5unit Klenow exo- in 1X Buffer? -> from buffer formula, tend to be Ampligase Buffer ? (total volume 10ul)
- - 25C 1h, heat inactivate 75C 20min
- - Add additional 10ul of BSPP in 1X Ampligase buffer (total volume 20ul)
- - Continue to BSPP capture, i) STD protocol or ii) multiple rounds of circularization
10X Exo-Minus Klenow DNA Polymerase Reaction Buffer (Epicentre): 200mM Tris-HCl (pH7.5), 50mM MgCl2, and 5.0mM DTT 10X Ampligase Reaction Buffer (Epicentre): 200mM Tris-HCl (pH8.3), 250mM KCl, 100mM MgCl2, 5mM NAD, and 1% Triton X-100
Capture program[edit]
1) BSPP capture, STD protocol[edit]
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. Note: In previous protocol, I used 100uM. I will change to 200uM to follow Deng et al, 2009)
2) BSPP capture, multiple rounds of circularization[edit]
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 4h -> 5X (95C 1min -> 55C 4h)
-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 4C hold.
- Continued on: [[1]]