Stock Number:015814
Strain Name:B6;CBA-Tg(Thy1-spH)64Vnmu/FrkJ
pHluorin
pHluorin
Separated PCR
1.0
25-April -2011 23-August -2011
Notes:

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

Have put internal controls on all 3 protocols. But only 1 set needs to be run if ithey are run separated.
Expected Results:

Transgene = 671 bp
Internal Control = 324 bp
Gel Image 
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Protocol Primers 
Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type
12274 - CTG GAG TTG TCC CAA TTC TTG TTG AAT TAG - Transgene Forward
12275 - GCC ATG TGT AAT CCC AGC AGC TGT TAC - Transgene Reverse
oIMR7338 - CTA GGC CAC AGA ATT GAA AGA TCT - -
oIMR7339 - GTA GGT GGA AAT TCT AGC ATC ATC C - -
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
2.5 mM dNTP 0.96 0.20 mM 0.96
DNA 2.00 0.00 2.00
ddH2O 4.92 - 4.92
5 mM DNA Loading Dye 1.66 0.69 mM 1.66
10 X KAPA Rxn Buffer 1.20 1.00 X 1.20
20 uM 12274 0.60 1.00 uM 0.60
20 uM 12275 0.60 1.00 uM 0.60
5 U/ul KAPATaq DNA Polymerase 0.06 0.03 U/ul 0.06
Cycling 
Step # Temp °C Time Note
1 94 3 min -
2 94 30sec -
3 64 30sec -
4 72 30sec -
5 - - Go to step 2-4, 35 times
6 72 2 min -
7 10 - hold
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
ddH2O 5.22 - 5.22
10 X KAPA Rxn Buffer 1.20 1.00 X 1.20
2.5 mM dNTP 0.96 0.20 mM 0.96
20 uM oIMR7338 0.45 0.75 uM 0.45
20 uM oIMR7339 0.45 0.75 uM 0.45
5 mM DNA Loading Dye 1.66 0.69 mM 1.66
5 U/ul KAPATaq DNA Polymerase 0.06 0.03 U/ul 0.06
DNA 2.00 - 2.00
This genotyping protocol is used for the following strains:
 
Stock Number Strain Name
015814 B6;CBA-Tg(Thy1-spH)64Vnmu/FrkJ
Genotyping resources and troubleshooting
This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.
Cycling 
Step # Temp °C Time Note
1 94 3 min -
2 94 30sec -
3 64 30sec -
4 72 30sec -
5 - - step 2-4,35 cycles
6 72 2 min -
7 10 0 Forever