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.
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