Protocol to Genotype Rs1 Mice:
R. 20100920
Note: DNA Extraction Method and PCR Reaction Mix of Sigma Aldrich as well as
Protocol are being use for genotyping. Product Code XNAT- 1KT. For detailed
Technical Bulletin go to sigma-aldrich.com.
Primers:
ECH009 TetORs1 F-1244: GCCCATCAGATCCAGATGTT
ECH004 TetORs1 R-1926: GGGTCCATGGTGATACAAGG
PCR product: 683 bp.
Reagents Needed (Provided in Sigma Kit)
For Tail Digest:
Extraction Solution Product Code E7526
Tissue Prep Solution Product Code T3073
Neutralization Solution B Prod. Code N3910
For PCR Reaction:
REDExtract-N-Amp PCR reaction Mix Product Code R4775
Procedure:
For DNA Extraction from tail tips:
1. Mix 50ul of Extraction Solution and 12.5ul of tissue prep solution
Note: If several reactions will be done, a master mix of extract
solution and tissue prep may be used (add 62.5 ul).
2. Add the previously cut 0.5 to 1 cm. Mouse tail tip to the solution.
3. Incubate sample at room temperature for 10 min.
4. Incubate sample at 95 C for 3 min.
5. Add 50ul of Neutralization solution B to sample and mix by
vortexing.
6. Store the neutralized tissue extract at 4 C or use immediately in PCR.
PCR amplification
1. Add the following reagents to a PCR tube:
Water (DI, or PCR grade)
RedExtract-N-Amp PCR mix
Forward Primer
Reverse Primer
Tissue extract
7.2
10.0
0.4
0.4
2.0
ul
ul
ul. (25 uM)
ul (25 uM)
ul
Total volume
20.0 ul
2. Mix all reagents gently.
3. Perform PCR in the following conditions: (program ED002)
1.
2.
3.
4.
5.
6.
7.
8.
94 C for 5:00
94 C for 1:00
58 C for 2:00
72 C for 2:00
Go to 2, 34 times
72 C for 15:00
15 C for ever
End
4. Analyze Product on a 2% agarose gel. Note: all 20ul of product may be
loaded directly to gel, no need to use a separate loading buffer.
Note:
1. Now using half the amount of Tail Digest reagents (to save reagents).
It used to be 100ul for Extract sol, and 25ul of Tissue prep. Results
have proven to be as effective.
2. Sigma Kit Protocol initially used only for Rs1/Col1 genotype.
However, was proven as a better PCR kit and method. It saves time
as well as it is cost effective. Now using the kit for every Conklin lab
mouse strain genotype. See Cost Analysis comparison.