Download New GFP and YFP Genotyping Protocol with BACE control (July

New GFP and YFP Genotyping Protocol with BACE control
(July 2012)
This protocol can be used to genotype flop-GluCl, TRE-GluCl, TRE-GlyCl, TetO-H2B-GFP, Ai3 cre reporter and
Thy1-GFP. These primers bind to sequences that are identical in GFP and YFP, and which are contained
entirely within the XFP cds (our former GFP protocol used a forward primer in XFP and a reverse in WPRE).
Reagent
ddH2O
10X PCR Buffer
25mM MgCl2
5mM dNTPs
New GFP F
New GFP R
Hc 69
Hc 70
Taq Polymerase
Tail DNA
Volume/rxn (ul)
37.7
5.0
1.5
1
0.2
0.2
0.2
0.2
1
3
PCR Program, Eppendorf Pro (Called “new GFP” under genotyping header)
1. 94C for 1.5 minutes
2. A. 94C for 30 seconds
B. 60C for 1 minute
C. 72C for 1 minute
D. 40 Cycles A-C
3. 72C for 2 minutes
4. Hold at 4C
Primer Sequences:
New GFP F: AAG TTC ATC TGC ACC ACC G
New GFP R: TCC TTG AAG AAG ATG GTG CG
Hc 69: AGG CAG CTT TGT GGA GAT GGT G
Hc 70: CGG GAA ATG GAA AGG CTA CTC C
Expected Products: Transgenic YFP or GFP
Transgenic animals for YFP or GFP should produce one band from the transgene at approximately 187kb.
Expected Products: BACE1 control
All animals, transgenic and wild-type, should exhibit the 272 kb band, indicating successful amplification of the
tail DNA.
Gel Percentage: 2%
Reference: Jackson Laboratory standard PCR for Generic GFP
http://jaxmice.jax.org/protocolsdb/f?p=116:2:973285084167622::NO:2:P2_MASTER_PROTOCOL_ID,P2_JRS_C
ODE:6125,003291