Sam:LabNotes/Human/2009-4-17
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Human MDA on L4N9, L4N8, L4N7 and N6 with Tre[edit]
Objective[edit]
- Exp. Design (total = 32 rxns)
Row A: Primer L4N9 30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3) Row B: Primer L4N8 30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3) Row C: Primer L4N7 30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3) Row D: Primer N6 30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3)
Preparation:[edit]
- Thaw all the reagents needed for MDA
- 10X buffer (from kit)
- Ambion RT-PCR H2O
- 1 mM DTT (Sigma)
- dNTP (from kit)
- N6 (1mM)
- pre-diluted gDNA standard (30pg, 3pg, 3 fg)
- Prepare fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
- 5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH. Ex. 0.093 g (KOH=56.11) = 0.001657 mole; 0.001657/5 x 10^6 =331.49 uL --- H2O needed
- Mix 200ul nuclease free H2O, 25ul 1M DTT(Sigma), 20ul 5M KOH, 5ul 0.5M EDTA
- Mix 10ul ALS buffer with 10ul NS buffer, check pH with a pH paper, adjust ALS buffer with KOH or H2O if pH is not within 7-8
- Extra-UV treatment for buffers and working solution-UV 15 min with the cap open
- RT-PCR grade H2O (Ambion)- 20 uL x 1 PCR tubes - for Blank
- ALS buffer - 20 uL x 4 PCR tubes (for 4 rows)
- NS buffer - 20 uL x 4 PCR tubes (for 4 rows)
- Set up Bio-Rad realtime PCR programing:
Line 1: 30 C, 6 min Line 2: Plate read Line 3: Goto line 1 for additional 99 times Line 4: 30 C, 6 min Line 5: Plate read Line 6: Goto line 1 for additional 99 times Line 7: 85 C, 3 min (inactivation) Line 8: 4 C, forever Line 9: End
- Prepare 2X SYBR (2 uL of 50X SYBR + 48 uL H2O)
- Prepare No-dNTP-MDA master mix (in a 1.5-mL tube)
1 rxn 9 rxns X 4 ------------------------------------------------------------- RT-PCR grade H2O 5.2 46.8 uL 10x RepliPhi phi-29 buffer 2.0 18.0 uL 1mM primer 1.0 9.0 uL - L4N9, L4N8, L4N7, N6 2X SYBR Green I 1.0 9.0 uL RepliPhi Phi-29 (100U/ul) 1.0 9.0 uL -------------------------------------------------------------- 10.2 91.8 uL (91.8/9=10.2) 25mM dNTP 0.8 uL 7.2 uL --- add dNTP when reactions are ready to set up
- Warm by the cool rack (from -20) at RT for 10 min
- Use a p200 pipettor to mix and pool the UVed buffer and H2O together
- Set up PCR tube strips on the rack at RT. Add 1.5 uL ALS into the tube.
- Transfer 1 uL of template into each of PCR tubes and mix by gently pipetting (4 times). Incubate at RT for 5 min (This step is still done at RT)
- Add 1.5 uL NS into the tube and mix by pipetting 4 times
- Add 5.0 uL Tre into the tube and mix by pipetting 4 times
!!! Add the dNTP into master mix !!!
- After add dNTP in phi29 master mix, transfer 11 uL master mix into each of tubes. Cap and label the tube. Mix by vortexing and quick spinning down.
- Place the reaction in Bio-Rad realtime PCR machine and start the reaction program (using 20-hr incubation setting).
- Inactivate all amplicons for 85C 3min
Results[edit]
File:041709 L4N9-tre.jpg File:041709 L4N8-tre.jpg File:041709 L4N7-tre.jpg File:041709 N6-tre.jpg
Discussion[edit]
- Performance (based on seperation between 30pg and NTC)
- L4N7>L4N8>L4N9>N6
- Although a whole new phi29 kit (enzyme + buffer) was used in this experiment, a 3fg-level contamination was found in most reactions. It is possible the contamination might exit in 50X SYBR (or primers ?)