Strain Protocols

Strain Information
Strain Code: 01ST2
Strain Name: B6.129S2(Cg)-Rb1tm1Tyj/Nci
Gene Symbol: Rb1
Protocol Information
Allele: Rb1<tm1Tyj>
Protocol Number: 1
Introductory Comment: This protocol uses a "generic" Neo primer set (N001 and N002) to detect the Neo cassette insertion. Primers N003 and N004 detect the endogenous mouse T-cell receptor delta (Tcrd) gene as an internal positive control (IPC), confirming the presence of amplifyable DNA in the reaction.

Primers

N001 : 5'-ctt ggg tgg aga ggc tat tc-3'
N003 : 5'-caa atg ttg ctt gtc tgg tg-3'
N002 : 5'-agg tga gat gac agg aga tc-3'
N004 : 5'-gtg agt cga gtg cac agt tt-3'

Product Sizes

Primer Combination Product Size
N001 / N002 280 bp    280 bp (Neo)
N003 / N004 200 bp    (Tcrd IPC)

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0 mM
2.5mM dNTP Mix 0.2 mM
100 µMN001 0.5 µM
100 µMN003 0.5 µM
100 µMN002 0.5 µM
100 µMN004 0.5 µM
5U/┬Ál Taq Pol 0.015 U
Use 50 - 100 ng of DNA per reaction

Cycling Conditions

(Time in minutes:seconds)
Temperature Time Label
94ºC 3:00
94ºC 1:00
60ºC 2:00 35 Cycles
72ºC 1:00
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.