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- Collect 500 µL of culture media. (Cells must be in culture for at least a couple of days).
- Boil 10 min at 95°C
- Spin 5 s at max speed
- Collect SN without disturbing the pellet
- Use 2 µL of this SN for PCR
- Set up 25µL reaction (recipe for DreamTaq, but any other Taq will do)
o 10x green buffer 2.5 µL
o dNTP (2.5 mM) 1.75
o mycUP primer (10 µM) 1.25 µL (ACT CCT ACG GGA GGC AGC AGT A)
o mycDW primer (10 µM) 1.25 µL (TGC ACC ATC TGT CAC TCT GTT AAC CTC)
o DreamTaq 0.25 µL
o Water 16.0 µL
+ (template) 2.0 µL
- Don't forget to include a positive control!
- Cycle:
a) 95°C, 1 min
b) 95°C, 30s
56°C, 30s
72°C, 45s repeat 34 times
c) 72°C, 10 min
- Load on 0.8% agarose gel
- Amplified band is about 720bp
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