HLATypingforA2.1TransgenicMice,protocol1

DNA Digestion and Extraction from Mouse Tail

v Cut about 5mm of mouse tail, place in a 1.6mL eppendorf tube on ice (if very cold, the tail is not going to stick to the teflon pestle) 
v Homogenize tails with sterile teflon pestle thoroughly 
v Add 100uL (or 200ul) Batteys digestion buffer (0.1M NaCl, 0.2M sucrose, 0.01M EDTA, 0.03M Tris, pH 8.0, and 0.4-1% SDS) 
v Add 5uL(or 10ul) proteinase K (20mg/mL stock) 
v Incubate at 55?C one hour, vortex every 15min 
v Add 17.5uL(or 35ul) of 8.0M potassium acetate, mix by vortexing, incubate on ice for one hour. 
v After incubation, spin down in cold room (@14k rpm 5-10min) and carefully remove the supernatant and transfer to a new tube.

*Do the following steps under the fume hood!!!!! *

Add 1 volume phenol and 1 volume of chloroform, vortex and spin @14k rpm 5min 
Take the upper phase, transfer to a new tube, add 1volume of chloroform, vortex and spin @ 14k rpm 5min 
Transfer supernatant into new tube 
Add 2.5 volumes freezer cold 100% ETOH (flick tube with finger to get the DNA to precipitate) 
Spin @14k rpm 20 min in cold room (@ 4?C), discard supernatant carefully (dont dislodge the pellet!!) 
Rinse pellet with 2.5.volumes of freezer cold 70% ETOH (finger flick to mix, spin down in cold room and discard supernatant) 
Let pellets air-dry 15-20min 
Add 30-60uL sterile water (or TE buffer) to dissolve DNA (pipet up and down to dissolve DNA).

Items needed:

Digestion buffer: (0.1M NaCl, 0.2M sucrose, 0.01M EDTA, 0.03M Tris, pH 8.0, and 0.4-1% SDS) 
Proteinase K: 20mg/mL stock 
Phenol 
Chloroform 
8M Potassium Acetate 
100% ETOH (stored in -20°C freezer) 
70% ETOH (stored in -20°C freezer) 
Sterile water (or TE buffer)

PCR of the HLA-A02 gene: ref Krausa et al , Tissue Antigens 1995: 45 , 223-231.

1)1st PCR: amplification product is 813bp.

primers AL#37: CCT CGT CCC CAG GCT CT 
primer AL#AW: TGG CCC CTG GTA CCC GT

DNA preparation: quantify DNA by OD reading and prepare 1ug/10ul for PCR reaction.

For 1 reaction: 50ul

10x Buffer 5ul 
dNTP's 4ul 
MgCL2 4ul 
Primers 2.5ul each 
TaqPol 0.5ul 
H2O 21.5ul 
DNA 1ug in 10ul H2O

Stock solutions:

10x buffer: 670mM Tris base pH 8.8, 166mM ammonium sulfate, 1% Tween 20 
dNTP's: mix volume/volume each of the 10mM dNTP. Final conc in PCR mix is 200uM each. 
MgCl2: stock is 25mM 
Primers: stock is 20uM

PCR cycles: program#139 in C209D.

1cycle: 94°C 3min 
5cycles: 96°C 25sec 
70°C 45sec 
72°C 30sec 
21cycles 96°C 25sec 
65°C 45sec 
72°C 30sec 
4cycles 96°C 25sec 
55°C 1min 
72°C 2min 
1cycle 72°C 10min 
1cycle 4°C forever

Run 10ul of the product on a 1% agarose gel.