Universal Mouse Genotyping Protocol

This protocol is designed to detect sequences in the murine genome by polymerase

chain reaction amplification, and is adapted from Stratman and Simon, Transgenic
Research 12, 521-522 (2003). Primers thirty nucleotides in length are picked that will
produce an amplimer from the sequence of interest. A single set of PCR conditions has
been developed that will produce an amplimer from any primer set. The only criteria are
that primers must contain approximately 50% guanosine or cytosine, and that the
amplimer be 100-400 nucleotides in length. More than one primer set may be included
in the reaction to detect multiple sequences, including amplimers that serve as internal
controls. DNA is prepared by briefly boiling a mouse toe or other tissue in base, then
adding a neutralization solution [Biotechniques 29(1), 52-54 (2000)], as described
below.

Primer Design

Primers must be picked that will amplify the sequence of interest but no other sequence.
Primers should be thirty nucleotides in length and contain 40-60% total guanosine or
cytosine residues. The primers should not have homologous three prime ends,
repetitive sequences, or long runs of a single residue. The amplimer should be 100-400
nucleotides in length (shorter amplimers usually work better). The table shows proven
primer pairs.

432bp amplimer length

GCA AAT GGG AGA GAC CCT TTG AAG TCA AGG
CTG GCA GAC CTT CTG TCT TCA TTT TCC AGG

400bp amplimer length

TAT CTA AAC AGA CTC CAC AGC CTC CAG ACC
CGA AGC CAG CTT GAG TTA CAG AAT GGG ATC

600bp amplimer length

GAC TGG GCA CAA CAG ACA ATC GGC TGC TCT
CGA AGC CAG CTT GAG TTA CAG AAT GGG ATC

389bp amplimer length

GTT GCA GTG CAC GGC AGA TAC ACT TGC TGA
GCC ACT GGT GTG GGC CAT AAT TCA ATT CGC

408bp amplimer length

GCA TTA CCG GTC GAT GCA ACG AGT GAT GAG
GAG TGA ACG AAC CTG GTC GAA ATC AGT GCG

194bp amplimer length

TGG ACA GGA CTG GAC CTC TGC TTT CCT AGA
TAG AGC TTT GCC ACA TCA CAG GTC ATT CAG

466bp amplimer length

CCT CCG GAG AGC AGC GAT TAA AAG TGT CAG
TAG AGC TTT GCC ACA TCA CAG GTC ATT CAG

323bp amplimer length

AGA CGC CAA AAA CAT AAA GAA AGG CCC GGC
TAT AAA TGT CGT TCG CGG GCG CAA CTG CAA

280bp amplimer length

GCA CGA CTT CTT CAA GTC CGC CAT GCC
GCG GAT CTT GAA GTT CAC CTT GAT GCC

360bp amplimer length

CTG CAC CAG CTG GCG TTT GAC ACC TAC CAG
TTT CTG TTG TGT TTC CTC CCT GTT GGA GGG

380bp amplimer length

TGC TCC TGC CGA GAA AGT ATC CAT CAT GGC
CGC CAA GCT CTT CAG CAA TAT CAC GGG TAG

335bp amplimer length

TGA GTG CTA GCT AGG CTT AGA GGT GCA AGC
AGT GGA TGG TGG TAT ACT CAG AGC CGG CCT

550bp amplimer length

CAT CGC CTT CTA TCG CCT TCT TGA CGA GTT
AGT GGA TGG TGG TAT ACT CAG AGC CGG CCT

190bp amplimer length

CTG CTA CAC AGT TGA CAT ACC TTA ATC TAC
AGA GCC TAG AGG TTA GGG ACA CAA CTC TTC

340bp amplimer length

TAA GGG AGT CCT GGT CTC TTT CTG TCT TTA
AGA GCC TAG AGG TTA GGG ACA CAA CTC TTC

The Fabpi pairs amplify a sequence from an endogenous murine gene and are suitable
for inclusion in any reaction as internal controls. he two Fabpi sets produce amplimers
of different lengths to allow inclusion one set with mutation-specific primers that produce
amplimers of any length. The Fabpi primers work with the other primers shown in the
table.

Sensitivity

This PCR genotyping assay has proven sensitive enough to detect genomic sequences
present as a single copy at one allele. This sensitivity is obviously necessary for
induced mutations such as gene knockouts, but is also important in identifying
transgenic founder animals that may be present as a single copy. We have compared
the sensitivity of the assay between plasmid standards and murine genomic DNA so
that assay sensitivity may be tested before generating mice containing the mutation. To
test sensitivity, make serial dilutions of your plasmid containing the mutation in water
containing mouse genomic DNA at a concentration of 500ng/µL. Plasmid
concentrations should be tenfold dilutions spanning 100pgµL to 0.01pg/µL. Set up the
standard PCR mix, using 1µl of each dilution as your DNA template. In order to assure
detection of single copy integrated transgenes, you need to be able to see a band at the
0.01pg level. Anything less sensitive may allow multiple copy detection, but it may be to
your advantage to test a different set of primers.

PCR reaction
Stock solutions are prepared ahead of time, and then used to assemble the working
master mixture. The master mixture is stable for at least two hours at room temperature
after all ingredients are added. The stock solutions can be made ahead of time and
stored at -20°C, except for the betaine which is kept at room temperature and the PCR
buffer which is stored at 4°C.

Reagent Stock Solutions

water
4M betaine in water Sigma B2754
10mM cresol red in water Sigma C9877
20mM dNTP mix in water 100µM dATP Promega U120A
100µM dGTP Promega U121A
100µM dCTP Promega U122A
100µM dTTP Promega U123A
10X KLA Buffer pH = 9.2 Wayne Barnes 314.362.3351
barnes@barnes1.wustl.edu
Klentaq LA (DNA polymerase)
20µM 5'-internal control primer in water
20µM 3'-internal control primer in water
20µM 5'-transgene primer in water
20µM 3'-transgene primer in water

Reaction Mixture

The total reaction volume is 20µL including the DNA sample, and cycling is done in thin-
walled tubes in a machine with a heated lid.

• Make a master mix containing the following reagents, where volumes listed are
for one reaction. You will need a master mix with enough reagents for the
number of samples and controls to be run, plus 10% extra for losses during
pipeting.

water 5.1 µL1 1

Adjust water so that reaction contains 20µL after
adding DNA template.
betaine 6.5 µL
10X buffer 2.0 µL

dNTP mix 0.05 µL

cresol Red 0.10 µL
5' Fabpi primer 0.12 µL2 2
Fabpi primers work best at this concentration -
3' Fabpi primer 0.12 µL 2 1.0µL should be used for all other primers.

5' transgene primer 1.0 µL

3' transgene primer 1.0 µL
Klentaq LA 0.04 µL
mouse DNA sample 4.0 µL

• Mix master mix well by flicking tube gently, then pulse spin in a microfuge.
• Aliquot 16µL of master mix into thin walled PCR tubes.
• Add 4µL of DNA sample to each tube.
• Place tubes in PCR machine.

PCR cycling

Run the following program:

• step 1. 93°C 1 minute

• step 2. 93°C 20 seconds
• step 3. 68°C 3 minutes
• repeat steps 2 and 3 for 30 cycles
• step 4. 4°C Hold

After the PCR reaction is finished, electropherese each entire PCR sample through a
2% agarose gel at 120V for approximately one hour.

HotSHOT preparation of murine DNA for PCR

This procedure is adapted from [Biotechniques 29(1), 52-54 (2000)]. Briefly, a single toe
is clipped from a mouse aged five days through adult. The toe is placed in a PCR tube
within one hour of cutting and may be stored frozen at -20°C. DNA is released from the
tissue by heating in a base solution, then adding a neutralization solution. The working
stocks can be made from the concentrate and used for two weeks.

Reagent Stock Solutions

base solution 50X stock = 1.25M NaOH, 10mM EDTA pH=12

• 250 mL 5N NaOH
• 20 mL 0.5M EDTA
• bring volume to 1L with water
 • Adjust pH to 12.0 with NaOH/HCl

base solution 1X working stock = 25mM NaOH, 0.2mM EDTA pH=12

• add 1mL of 50X stock solution to 49mL water

neutralization solution 50X stock = 2M Tris-HCl pH=5

• 315.2g Tris HCl

• bring volume to 1L with water
• adjust pH to 5.0 with NaOH/HCl

neutralization solution 1X working stock = 40mM Tris-HCl pH=5

• add 1mL of 50X stock and 49mL water

Protocol

• Cut toe and place in a thin-walled PCR tube, and freeze until ready to elute DNA.
• Add 75µl of base solution 1X working stock to each sample.
• Place tubes at 95°C in a PCR machine (or bath or block) for 30 minutes.
• Cool to room temperature or below.
• Add 75µl neutralization solution 1X working stock and vortex.
• Use 4µl of this eluate in a 20µl PCR reaction.