Strain Protocols

Strain Information
Strain Code: 01XP1
Strain Name: B6;129-Polhtm1Rak/Nci
Gene Symbol: Polh
Protocol Information
Allele: Polh<tm1.1Rak>
Protocol Number: 1
Introductory Comment: DNA Polymerase eta (Polh, XPV) was inactivated by Cre-loxP mediated germline deletion of exon 4. The PCR strategy is to detect the absence of Exon 4 with primers XPV-mut and XPV-com. This strain is maintained by homozygous sibling intercrossing. However, hemizygotes can be distinguished from homozygotes if need be by the addition of primer XPV-wt.

Primers

com : 5'-aag gga caa gcg aac aga ga-3'
wt : 5'-tca ctt caa cac tag ctt ccc-3'
mut : 5'-agc aat atc aca ggc cca ac-3'
Diagram showing the primer relataionships for Strain 01XP1.  DNA Polymerase eta (Polh, XPV) was inactivated by Cre-loxP mediated germline deletion of exon 4.   The PCR strategy is to detect the absence of Exon 4 with primers XPV-mut and XPV-com.  This strain is maintained by homozygous sibling intercrossing.  However, hemizygotes can be distinguished from homozygotes if need be by the addition of primer XPV-wt.

Product Sizes

Primer Combination Product Size
com / wt 370 bp    WT
com / mut 500 bp    Mut

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0mM
2.5mM dNTP Mix 0.2mM
100 µMcom 0.5 µM
100 µMwt 0.5 µM
100 µMmut 0.5 µM
5U/┬Ál Taq Pol 0.025 U/µl
Use 50 - 100 ng of DNA per reaction

Cycling Conditions

(Time in minutes:seconds)
Temperature Time Label
94ºC 3:00
94ºC 0:30
60ºC 0:30 35 Cycles
72ºC 0:30
72ºC 3:00
4ºC HOLD

Disclaimer: PCR conditions were optimized using QIAGEN-purified mouse tail DNA and a PTC-200 thermocycler (MJ Research). Optimal conditions may vary with DNA quality or equipment used.