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. 

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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

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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.