Strain Protocols

Strain Information
Strain Code: 01XBH
Strain Name: B6.129-Tg(HHV8ORF73,Tk-GFP)1Dtmr/Nci
Gene Symbol: LANA
Protocol Information
Allele: Tg(HHV8ORF73, Tk-GFP)1Dtmr
Protocol Number: 1
Introductory Comment: This strain contains a transgene comprised of the latency-associated nuclear antigen (LANA) gene from Human Herpes Virus 8 under the control of its own promoter. Additionally, in an attempt to reduce epigenetic inactivation, the TK promoter was included downstream of the LANA coding region. The genotyping strategy is to detect the LANA coding region with primers LANA78-F and LANA78-R. They will produce an amplicon that is ~200bp long, representing transgene carriers.

Primers

LANAF : 5'-gga aga gcc cat aat ctt gc-3'
LANAR : 5'-gcc tca tac gaa ctc cag gt-3'
Diagram showing the primer relataionships for Strain 01XBH.  This strain contains a transgene comprised of the latency-associated nuclear antigen (LANA) gene from Human Herpes Virus 8 under the control of its own promoter.  Additionally, in an attempt to reduce epigenetic inactivation, the TK promoter was included downstream of the LANA coding region.  The genotyping strategy is to detect the LANA coding region with primers LANA78-F and LANA78-R.  They will produce an amplicon that is ~200bp long, representing transgene carriers.

Product Sizes

Primer Combination Product Size
LANAF / LANAR ~200bp    Tg

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0mM
2.5mM dNTP Mix 0.2mM
100 µMLANAF 0.5 µM
100 µMLANAR 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.