PCR Protocol for genotyping Insm1.GFP-Cre.Hygro mice
Kathy Shelton
The following conditions are used by the Magnuson Lab to genotype mice derived from a
RMCE event at the Insm1 gene targeted locus. Of the events pictured below, genotyping
reactions for Insm1wt and Insm1GFP-Cre.Hygro(+ or -) apply.
PCR screening of Insm1 gene targeting & RMCE events
2500
5000
7500
10000
12500
15000
1
Insm1wt
4
1
6
puTK/EM7neo
LA
5
SA
4
1
Primers:
1. Insm1.5a
2. Insm1.5b
3. F-Insm1
4. R-Insm1
5. pdTK5’
6. EMneo5b
7. Hygro 3’
SA
Target region
2
Insm1GFP-Cre.Hygro
20000
3
LA
Insm1targeted
17500
3
LA
Insm1
2
GFP-Cre Insm1
7
Hyg
SA
4
wt & RMCE with and without hygro screenings:
1 + 2 => 362 bp (wt) & 423 bp (Insm1GFP-Cre.Hygro & Insm1GFP-Cre.Hygro-)
3 + 4 => 449 bp (wt) & 561 bp (Insm1GFP-Cre.Hygro-)
Targeted allele screenings:
1 + 5 => 514 bp (targeted)
4 + 6 => 475 bp (targeted)
RMCE screenings for Hygro:
4 + 7 => 567 bp (Insm1GFP-Cre.Hygro)
PCR reagents:
1. oligo primers at 20 µM
Insm1 genotyping primers (see combinations and resulting amplicon sizes
noted in above figure)
Insm1.5a
5’- ATCCTCAGATTGTACTCAATACCTA-3’ (top)
Insm1.5b
5’- CCTGCATGTCCACACTGCGAT-3’ (bottom)
F-Insm1
5’- GCCCTTGTACAACCGACAGCTCT-3’ (top)
R-Insm1
5’- GTGCCCTGTATCTGCTGTGCAGA-3’ (bottom)
Hygro 3’
5’- GTGAGAACAGAGTACCTACAT-3’ (top)
Internal control primers give a 150 bp product in all genomic DNA samples
oMIR015
5’-CAA ATG TTG CTT GTC TGG TG-3’
oMIR016
5’-GTC AGT CGA GTG CAC AGT TT-3’
The PCR conditions outlined here are optimum for no more than two
different fragment amplifications per reaction. Hence, the internal control
primers can only be used in the hygro screening reactions.
2.
3.
4.
5.
Flpe screening is necessary once Insm1 animals have been crossed with this
line to facilitate the deletion of the flrted (flanked by FRT sites) pgk-Hygro
selection cassette include in the exchange construct for ES cell selection
following RMCE electroporation. See separate PCR protocol for this
genotyping reaction.
Perkin Elmer PCR buffer with MgCl2
dNTP premix (I make my own dNTP premix using 100 mM NEB dNTP’s.
The premix contains 250 µl of each dNTP - A, C, G, &T - and 19 ml sterile
water and is stable at -20°C. I freeze this in 1 ml aliquots and thaw and
refreeze as needed.)
Perkin Elmer Amplitaq Gold
genomic DNA samples diluted to 50 ng/µl with sterile water
PCR reaction mixture:
15.8 µl sterile water
2.5 µl 10X PCR buffer
4 µl dNTP premix
0.75 µl primer #1
0.75 µl primer #2
1 µl dil. DNA template
0.2 µl Amplitaq Gold
25 ul total volume
Cycling conditions:
1 cycle - 94°Cx 6 min.
40 cycles - 94°C x 1 min., 60°C x 30 sec., 72°C x 30 sec.
1 cycle - 72°C x 7 min.
hold at 4°C.
Analysis of PCR products:
Load 10 µl aliquots of reactions + 2 µl gel loading buffer in a 1% mini-agarose gel (using
a 15-well comb). Run gel approximately half-way down.
Sample gels illustrate amplicons for:
1. Insm1 5’-screenings (Insm1.5a + Insm1.5b)
2. Insm1 3’-screenings (F-Insm1 + R-Insm1 to detect wt and hygro deleted
samples)
3. Hygro screenings (Hygro 3’ + R-Insm1 to determine if Hygro is present)
Standards = Hind III digested DNA + Hae III digested PhiX 174 DNA
Genotypes deduced from above PCR gels were as follows, Flpe PCR screening not
shown:
6. Insm1.GFP-Cre(hygro+)/w
15. Insm1.GFP-Cre(hygro+)/w
16. Insm1.GFP-Cre(hygro-)/w.Flpe/w
17. w/w
18. Insm1.GFP-Cre(hygro-)/w.Flpe/w
19. Insm1.GFP-Cre(hygro-)/w.Flpe/w
20. w/w.Flpe/w
21. Insm1.GFP-Cre(hygro-)/w.Flpe/w
22. w/w.Flpe/w
23. w/w
24. w/w
25. w/w
26. w/w