SYBRGreen Q-PCR in the ABI 7700
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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)
KG-1 (10x) | KG-2 (10x) | |||
Amount | [final] | Amount | [final] | |
8.3 mL 1M (NH4 )2 SO4 | 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 H2 O | ||
q.s. to 50 mL with H2 O |
1. Create a master mix by multiplying this recipe by the number of reactions (add 10% for pipeting error):
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 H2 O
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)
Reference = Rox (unless no reference is used)
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...