Protocol P.4

PCR Genotyping from Tail DNA

This protocol works well for a variety of genes and primer pairs including Tg and KO alleles. Oligonucleotide melting temperatures between 60° and 65° seem to work well.

 

Solutions

HMW Lysis Mixture

10 mM Tris 8.5 1 ml 1M Tris pH 8.5

5 mM EDTA 8.0 1 ml 0.5M EDTA pH 8.0

0.2% SDS 1 ml 20% SDS

200 mM NaCl 4 ml 5M NaCl

up to 100 ml with Q

To use add 100 microliters of Proteinase K (10 mg/ml) for every 10 ml.

10X PCR Buffer 1

166 mM Ammonium Sulfate 8.3 ml 1M (NH4)2SO4

670 mM Tris 8.8 33.5 ml 1M Tris pH 8.8

67 mM EDTA 8.0 6.7 ml 0.5 M EDTA pH 8.0

67 mM MgCl2 3.35 ml 1M MgCl2

add 174 ml bME and bring up to 50 ml with Q, aliquote and store at -80°C

10X PCR Buffer 2

10 mM dGTP 25 ml 100 mM dGTP

10 mM dATP 25 ml 100 mM dATP

10 mM dCTP 25 ml 100 mM dCTP

10 mM dTTP 25 ml 100 mM dTTP

10% DMSO 25 ml DMSO

0.8 mg/ml BSA 20 ml 10 mg/ml BSA

105 ml Q

aliquote ant store at -80°C

 

 

 

 

 

Procedure

chromosomal dna preparation

• Clip 0.5-1 cm of tail and place in a sterile eppendorf tube, proceed immediately or store at -20°C.

• Add 500 microliters of HMW Lysis Mixture and incubate at 55°C overnight.

• Phenol/CHCl3 extract twice and CHCl3 extract once.

• Add 1/25 volume (16 ml) 5M NaCl and 2 volumes (0.8 ml) of EtOH.

Invert several times until a white ppt. forms and then quickly spin in the microfuge. Wash with 80% EtOH and air dry for 5-10 minutes.

• Add 0.5 ml TE and store at 4°C.

 

pcr genotyping

• Add 2 ml chromosomal DNA to each tube and 1 ml of each primer.

• Make a cocktail for the appropriate number of tubes as follows:

4 ml 10X PCR buffer 1

4 ml 10X PCR buffer 2

0.02 ml recombinant Taq (see protocol T.2)

28 ml sterile Q

• Add 36 ml per tube and perform PCR using the following conditions:

(96° for 30 seconds, 57° for 30 seconds, 65° for 2 minutes) x 4

(93° for 30 seconds, 57° for 30 seconds, 65° for 2 minutes) x 36

(65° for 2 minutes, 4° hold)