Col1a1 3'UTR mutation 2
Col1a1 3'UTR assay2 (flip-in)
Separated PCR
1.0
20-September-2011 20-September-2011
Expected Results:

HET = 331 bp and ~500 bp
MUT = ~500 bp
WT = 331 bp 
Gel Image 
Image Image file
View Full Size
Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.
Protocol Primers 
Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type
oIMR6724 - CCC TCC ATG TGT GAC CAA GG - Common
oIMR6725 - GCA CAG CAT TGC GGA CAT GC - Wild type Forward
oIMR6726 - GCA GAA GCG CGG CCG TCT GG - Mutant Forward
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
ddH2O 3.96 - 3.96
10 X AB PCR BufferII 1.20 1.00 X 1.20
25 mM MgCl2 0.96 2.00 mM 0.96
2.5 mM dNTP 0.96 0.20 mM 0.96
20 uM oIMR6724 0.60 1.00 uM 0.60
20 uM oIMR6726 0.60 1.00 uM 0.60
5 mM DNA Loading Dye 1.66 0.69 mM 1.66
5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06
DNA 2.00 - 2.00
Cycling 
Step # Temp °C Time Note
1 94 3 min -
2 94 30 sec -
3 56 30 sec -
4 72 30 sec repeat steps 2-4 for 35 cycles
5 72 2 min -
6 10 - hold
Reaction/Components 
Reaction Component Volume (µl) Final
Concentration		 Total Volume (µl)
ddH2O 3.96 - 3.96
10 X AB PCR BufferII 1.20 1.00 X 1.20
25 mM MgCl2 0.96 2.00 mM 0.96
2.5 mM dNTP 0.96 0.20 mM 0.96
20 uM oIMR6724 0.60 1.00 uM 0.60
20 uM oIMR6725 0.60 1.00 uM 0.60
5 mM DNA Loading Dye 1.66 0.69 mM 1.66
5 U/ul Taq 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
006911 B6;129-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm2(tetO-Pou5f1)Jae/J
011001 B6;129S4-Col1a1tm1(tetO-Pou5f1,-Klf4,-Sox2,-Myc)Hoch/J
014588 B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1A1tm6(tetO-MSI2)Jae/J
014592 B6;129S4-Col1a1tm1(tetO-mCherry)Eggn/J
014602 B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm1(tetO-mCherry)Eggn/J
016836 B6;129S4-Gt(ROSA)26Sortm1(rtTA*M2)Jae Col1a1tm7(tetO-HIST1H2BJ/GFP)Jae/J
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 30 sec -
3 56 30 sec -
4 72 30 sec repeat steps 2-4 for 35 cycles
5 72 2 min -
6 10 - hold