Dinh/NOTES/2010-1-7
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RT PCR of Bisulfite Converted DNA[edit]
Reaction Mix:
Reagent | Volume (1x) | 3x13 + 3 = 42x |
2x iQ Supermix | 20 | 840 |
Q-primer (25uM) | 2 | 84 |
BS converted gDNA | 0.3 | x3 = 0.9 |
dH20 | 17.7 | 743.4 |
119.1 ul master mix + 0.9 template = 120 ul -> 4 reactions / 30ul per reaction.
row 1 | NTC(M-Elution Buffer) | NTC(dH20) |
row 2 | Bis_10 | Bis_11 |
row 3 | Bis_12 | Bis_13 |
row 4 | Bis_14 | Bis_15 |
row 5 | Bis_16 | Bis_17 |
row 6 | Bis_18 | Bis_19 |
row 7 | Jurkat (1/6/07) | BS Jurkat gDNA (285ng/ul) |
Program:
96C (3min) -> (40 cycles: 95C (30s)->62C (45s)->72C (45s) -> plate read) -> 72C (5min) -> 15C forever
Results[edit]
ng/ul | Bis_10 | Bis_11 | Bis_12 | Bis_13 | Bis_14 | Bis_15 | Bis_16 | Bis_17 | Bis_18 | Bis_19 |
calc from Jurkat 1 | 578.54 | 756.37 | 805.62 | 1221.93 | 799.13 | 691.83 | 660.44 | 731.96 | ||
calc from Jurkat 2 | 125.71 | 164.34 | 175.04 | 265.5 | 173.63 | 150.32 | 143.5 | 159.04 | ||
forecast | 277.96 | 304.03 | 310.16 | 350.67 | 309.37 | 295.35 | 290.84 | 300.83 | ||
RT PCR of Capture Products (optimization)[edit]
PCR Amplification
Reagent | Vol (1x) | Vol (11x) |
Template (*) | 10ul | |
2x iProof MM | 50ul | 550ul |
AmpF6.2Sol (10uM) | 4ul | 44ul |
AmpR6.2Sol (10uM) | 4ul | 44ul |
50x SYBR Green | 0.8ul | 8.8ul |
RNase Free H20 | 31.2ul | 343.2ul |
Total | 100ul |
(*) Templates: Strip 1:
1. NTC 2. 0 Cycle - NTC - A 3. 0 Cycle A 4. 0 Cycle B 5. 4 Cycles - NTC - B 6. 4 Cycles - C1 7. 4 Cycles - C2
Strip 2:
8. 6 Cycles - NTC - B 9. 6 Cycles - A 10. 6 Cycles - B
Program
98C -> 30s 98C -> 10s 58C -> 20s 72C -> 20s Plate Read Go to 2 for 7x 98C -> 10s 72C -> 20s Plate Read Go to 7 for 15x 72C -> 3min 15C -> forever End
Result[edit]
Total of 24 cycles.
Conditions: A: 0.5mM dNTP in SLN mix B: 0.2mM dNTP in SLN mix C: 0.2mM dNTP in 1st SLN mix and and 0.5mM dNTP in 2nd
Discussion[edit]
The expected capture product appears at ~390 bp. Unspecific captured appears at 200bp or less. Zero cycling gives the best result and least amount of unspecific capturing. Fewer PCR cycles might be necessary to reduce over-amplification or duplicate reads.