SYBRGreen Q-PCR in the ABI 7700
Materials
ROX Passive Reference Dye 25 µM, 50x (Invitrogen, Cat. No. 12223-012)
SYBR Green I, "10,000x" concentrate (Molecular Probes, Cat. No. S7563)
SureStart Taq polymerase 5 U/µL (Stratagene, Cat. No. 600280)
Solutions
SYBR Green 1:100 dilution (50 µL SYBR Green concentrate, 5 mL DMSO, store aliquots at -20ºC in dark tubes in a dessicator)
SYBR Green 1:2000 solution (0.5 mL of SYBR Green 1:100 dil., 1 mL glycerol, 10 uL Tween, 8.5 mL H2O, store at -20ºC in dark tubes)
| KG-1 (10x) | KG-2 (10x) | |||
| Amount | [final] | Amount | [final] | |
| 8.3 mL 1M (NH4)2SO4 | 166mM | 50 µL of 100mM dNTP Stocks (x4) | (@10 mM) | |
| 33.5 mL Tris pH 8.8 | 670 mM | 50 µL DMSO | 10% | |
| 174 µL B-ME | 50 mM | 50 µL BSA (8 mg/mL stock) | 0.8 mg/mL | |
| 3.35 mL 1M MgCl2 | 67 mM | 200 µL H2O | ||
| q.s. to 50 mL with H2O |
PCR Reaction Setup
1. Create a master mix by multiplying this recipe by the number of reactions (add 10% for pipeting error):
2 µL Primer-1
2 µL Primer-2
2 µL KG-1
2 µL KG-2
5 µL 1:2000 SYBR Green solution
0.4 µL ROX Dye 25 µM
0.1 µL SureStart Taq
6.5 µL H2O
2. Setup individual reactions in PCR tubes or plates with optical caps or optical tape:
20 µL Master Mix
1 µL DNA (~ 50 ng genomic DNA)
Machine Setup
1. If the machine status reads "Idle" but a user still has not remove their samples then "Save" the current file to preserve the users data before shutting down.
2. Shut down both the machine and the computer. Turn on the detector first then boot the computer.
3. Launch SDS v1.9 software (don't use v1.7).
4. Create a new template:
Dye Layer = SybrGreen
Sample Type = Sample type setup. Choose SYBRG for UNKN, NTC. Deselect quencher.
Select wells with samples. Set sample type to UNKN (or NTC).
5. Program thermocycle conditions
95ºC 5' Hold (Stage I)
95ºC 15", 60ºC 30", 72ºC 30" (x40) (Stage II - this stage is emperic)
60ºC 15" Hold (Stage III)
95ºC 15" Hold (Stage IV)
Select Stage IV and set ramp time to maximum (19:59).
Select "Show Data Collection..."
Select and delete the documents for stage III and IV.
Select to add documentation of the Stage IV ramp.
6. Select "Show Analsis..." > Instrument > Diagnostics > Advanced Options
Select: Set 7700 exposure time for plates 25 (caps), or 10 (film).
Reference = Rox (unless no reference is used)
7. Save the template to the "Fero Lab" folder.
8. Place plate in sample block, close lid and shut door. Select "RUN".
Saving and processing data
9. When the machine has finished cycling the status will read, "Idle". Save the file again before quitting.
10. To save dissociation curves: select File > Export... > Multicomponent...
