PCR
From CCGB
PCR protocol
-- by Hao Wang
To amplify pre-CRM from genomic DNA, use Advantage 2 PCR Enzyme System, to screen by PCR, use Taq pol from GeneChoice.
1.Make up PCR master mix as follows
PCR buffer 1X
dNTP 200μM (each)
Forward_Primer 1μM
Reverse_Primer 1μM
DMSO 10 μl
Advantage 2 2.5μl
Total 240μl (made up in sterile ddH2O)
Master mix for 10 reactions 8 reactions
PCR buffer (10X) 25μl 20
dNTP (10mM) 5μl 4
Forward_Primer (50μM) 5μl 4
Reverse_Primer (50μM) 5μl 4
DMSO 10μl 8
Advantage 2 2.5μl 2
H2O 187.5μl 150
Total 240μl 192
2. Add 24 μl of PCR reaction mix to 0.6 ml Eppendorf tube.
Add 1 μl of genomic DNA or RT as template.
3. Run PCR reaction for 35 cycles (genomic DNA as template)
Use the following thermocycle
94°C, 2min→35×[94°C,1min→Ta, 1min→72°C, 1min]→72°C, 10min→7°C
or 25-30 cycles (RT or cell suspension as template)
Use the following thermocycle
94°C, 2min→25×[94°C,30sec→Ta, 30sec→72°C, 1min]→7°C
PCR screening of colonies
1. Chosse PCR primers to use in PCR screening.
2. Prepare aliquots of 40 μl water in Eppendorf tubes (one for each colony to be screened). 3-5 colonies from each vector+insert transformation plate are usually sufficient.
3. Use sterile loop to transfer single colonies to 40 μl aliquots of water (swirl loop in water). Re-streak the loop after swirling onto appropriate media plate (usually LB+amp).
4. Vortex the cell suspensions for 5 seconds. The cell suspension can be kept for at least 6 hours at room temperature before use.
5. Prepare PCR reaction mix
6. Add 19 μl of PCR reaction mix to 0.6 ml Eppendorf tube. Add 1 μl of cell suspension.
7. PCR amplify samples with 25-30 cycles in thermocycler following:
94°C, 2min→25×[94°C,30sec→Ta, 30sec→72°C, 1min]→7°C
