Universal Mouse Genotyping Protocol

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 [email protected]

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 thinwalled 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

betaine

6.5 µL

10X buffer

2.0 µL

dNTP mix

0.05 µL

1

Adjust water so that reaction contains 20µL after adding DNA template.

cresol Red

0.10 µL

5' Fabpi primer

0.12 µL2

3' Fabpi primer

0.12 µL

2

5' transgene primer

1.0 µL

3' transgene primer

1.0 µL

Klentaq LA

0.04 µL

mouse DNA sample

4.0 µL

• • • •

2

Fabpi primers work best at this concentration 1.0µL should be used for all other primers.

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.