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Tempel Lab Protocols

GFAP-GFP GENOTYPE

You will Need

Mouse Tail Digests

4 Primers:
WTF42
WTR43
MTF872
MTR1416

PCR Reagents:
Sterile water
10x buffer w/ MgCl2
10mM Nucleotide mix
FastStart Taq

Primer Sequences

WTF42: CTAGGCCACAGAATTGAAAGATCT
WTR43: GTAGGTGGAAATTCTAGCATCATCC
MTF872: AAGTTCATCTGCACCACCG
MTR1416: TCCTTGAAGAAGATGGTGCG

Procedure

Run the wildtype (WTF42 & WTR43 primers) and the mutant (GFP) (MTF872 & MTR1416 primers) PCR reactions in seperate tubes.

PCR Rxn

Sterile Water

17.8 ul

10x PCR buffer w/MgCl2

2.5ul

10mM Nucleotide mix

0.5ul

Common Forward primer 20 uM

1ul

Reverse primer 20 uM: WT Rev or MT Rev

1ul

FastStart Taq

0.2ul

Tail Lysate

2ul

Total Volume

25ul
   
   
   
 

PCR Protocol

94 deg C

4:30

  

94 deg C

0:30

  

65 deg C

1:00

x35

72 deg C

1:00

  

72 deg C

7:00

  

4 deg C

Hold

  
     

Gel
1.5 % Agarose + 2 ul EtBr (10mg/ml)

Bands
Wildtype (GFP-): 324 bp internal control
Mutant (GFP+): 173bp