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Universal GENSAT PCR Protocol



MMRRC's Universal PCR Protocol for GENSAT EGFP Transgenes

In combination with the correct forward primer, this protocol can be used to confirm the presence of the particular transgene construct.

NOTE:
Locate your promoter gene in GENSAT's A-Box Primers Table to obtain the primer sequence used to create the A-box of the transgene construct. You will need to use the reverse-compliment of this sequence, adjusted in length to accommodate the melting/annealing temperatures of the given GFP R2 primer.

Reagents:

Reagent / Constituent Concentration Volume (µL)
buffer (without MgCl2) 10X 2.5
MgCl2 (concentration may be adjusted to reduce non-specific banding) 25 mM 1.0
dNTPs 10 mM 0.5
Primer 1 Name: strain-specific forward primer 10 µM 0.5
Primer 2 Name: EGFP reverse primer 10 µM 0.5
Taq Polymerase 5 Units / µl  0.5
DNA sample 1.0
Water 18.5
Total Volume of Reaction: 25.0 µL


Primers:

# Primer Name Nucleotide Sequence (5' - 3')
1 strain specific forward primer length-adjusted reverse-compliment of your A-box primer sequence from GENSAT's A-Box Primers Table
2 GFP R2 TAg Cgg CTg AAg CAC TgC A


Cycles:

Step Temp. (°C) Delta per cycle Time(m:ss.) # of Cycles
1. Initial melting 95° - 5:00 1
2. Denaturization 94° - 0:20 - 
3. Anealing 60° to 50° decrease 0.5° per cycle for 10 cycles; next 10 cycles at 55° last 15 cycles at 50° 0:30 - 
4. Elongation 72° - 0:30 - 
Repeat steps 2, 3, 4 total cycles: 35
5. Amplification 72° - 5:00 1
Finish 25° - Hold n/a

 

Electrophoresis: Sample Gel: Lane key:
2%  Agarose gel at 90 Volts for 90 mins. Sample Gel 
   1: 1 kb+ ladder (Invitrogen, Cat. # 10787-026)
   2: H2O
   3: Wild Type
   4 - 6: strain specific Tg/+
Band Interpretation:
Band # Position Genotype
1 None Wild Type
2 ~ 300 bp Transgene

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Last Modified: December 19, 2005