Stock Number: 005026 Strain Name: FVB.Cg-Tg(SMN2)89Ahmb Tg(SMN1*A2G)2023Ahmb Smn1tm1Msd/J

Allele: Tg(SMN2)89Ahmb Protocol Name: Tg(SMN2)89Ahmb Method: Melt Curve Analysis Version: 1.1 Created: 04-February -2009 Updated: 16-May -2012

Notes: Melting curve analysis is done using a Roche Light Cycler 480. This assay DOES distinguish hemizygous from homozygous transgenic animals.  Specific hemizygous and homozygous controls should be used. Expected Results: Transgene Tm = 79C +/- 1.0°C Internal positive control Tm = 79°C+/- 1.0°C

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 oIMR5065 - CTG ACC TAC CAG GGA TGA GG - Common oIMR5066 - GGT CTG TTC TAC AGC CAC AGC - Transgene oIMR5067 - CCC AGG TGG TTT ATA GAC TCA GA - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.80 - 2.80 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR5065 0.60 1.00 uM 0.60 20 uM oIMR5066 0.60 1.00 uM 0.60 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 5 min - 2 94 30 sec - 3 70 1 min - 4 72 45 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 2.80 - 2.80 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR5065 0.60 1.00 uM 0.60 20 uM oIMR5067 0.60 1.00 uM 0.60 DNA 2.00 - 2.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 005024 FVB.Cg-Tg(SMN2)89Ahmb Smn1tm1Msd/J 005025 FVB.Cg-Tg(SMN2*delta7)4299Ahmb Tg(SMN2)89Ahmb Smn1tm1Msd/J 005026 FVB.Cg-Tg(SMN2)89Ahmb Tg(SMN1*A2G)2023Ahmb Smn1tm1Msd/J 006570 STOCK Smn1tm1Msd Tg(Hlxb9-GFP)1Tmj Tg(SMN2)89Ahmb/J 007951 STOCK Smn1tm3(SMN2/Smn1)Mrph Tg(SMN2*delta7)4299Ahmb Tg(SMN2)89Ahmb/J 008203 STOCK Smn1tm1Msd Tg(ACTA1-SMN)63Ahmb Tg(SMN2)89Ahmb/J 008209 FVB.Cg-Smn1tm1Msd Tg(ACTA1-SMN)69Ahmb Tg(SMN2)89Ahmb/J 008212 STOCK Smn1tm1Msd Tg(Prnp-SMN)92Ahmb Tg(SMN2)89Ahmb/J 008782 FVB.Cg-Smn1tm1Msd Tg(SMN2)89Ahmb Tg(SMN2*A111G)588Ahmb/J 009134 FVB.Cg-Smn1tm1Msd Tg(SMN2)89Ahmb Tg(SMN2*A111G)591Ahmb/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  None found.