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PCR Protocol

EGFP Genotyping Protocol at Donor's Laboratory for GENSAT BAC transgenic strains


Reagents:

Reagent / Constituent Concentration Volume (µL)
buffer (Qiagen) 10X 2.0
MgCl2 (Qiagen) 15 mM 0.8
dNTPs (Qiagen) 2.5mM 1.6
Primer 1 Name: EGFP forward primer 50 µM 0.2
Primer 2 Name: EGFP reverse primer 50 µM 0.2
Primer 3 Name: Actin PCR control/forward 50 µM 0.4
Primer 4 Name: Actin PCR control/reverse 50 µM 0.4
Tag Polymerase (Qiagen)   0.2
Q solution (Qiagen) 4.0
Water 9.2
tail genomic DNA sample 1.0
Total Volume of Reaction: 20.0 µL


Primers:

# Primer Name Nucleotide Sequence (5' - 3')
1 EGFP forward primer CCT ACG GCG TGC AGT GCT TCA GC
2 EGFP reverse primer CGG CGA GCT GCA CGC TGC GTC CTC
3 Actin PCR control/forward primer GAT GAC GAT ATC GCT GCG CTG GTC G
4 Actin PCR control/reverse primer GCC TGT GGT ACG ACC AGA GGC ATA CAG


Cycles:

Step Temp. (°C) Delta per cycle Time(secs.) # of Cycles
1. Initial melting 94 - 180 1
2. Denaturization 94 - 30 - 
3. Anealing 60 - 45 - 
4. Elongation 72 - 45 - 
Repeat steps 2, 3, 4 total cycles: 30
5. Amplification 72 - 600 1
Finish 4 - indefinite n/a


Electrophoresis:  

2.5% Agarose gel at 200-250 Volts for 30-45 mins.

Band # Position Genotype
1 ~300 bp EGFP
2 1 kb Actin (control)



Last Modified: April 04, 2005

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Last Modified: September 15, 2008