HLATypingforA2.1TransgenicMice,protocol2

2) Nested PCR: amplification product is 715 bp.

Primer AL#22: CAC TCC ATG AGG TAT TTC TT 
Primer AL#Q: CTC TCT GCT GCT CCG CCA

DNA preparation: make a 1:100 dilution of the first PCR (for example 5ul PCR product into 495ul H2O). Add 5ul into nested PCR mix.

For 1 reaction: 50ul

10x Buffer 5ul 
dNTP's 4ul 
MgCL2 4ul 
Primers 2.5ul each 
TaqPol 0.5ul 
H2O 26.5ul 
DNA 5ul of previous 1/100 reaction

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#134 in C209D.

1cycle: 94°C 3min 
30cycles 94°C 45sec 
55°C 45sec 
72°C 2min 
1cycle 4°C forever

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

PCR of the housekeeping gene G3PDH: ref Clontech

Primer 5': ACC ACA GTC CAT GCC ATC AC 
Primer 3': TCC ACC ACC CTG TTG CTG TA

DNA preparation: quantify DNA by OD reading and prepare 1ug/10ul for PCR reaction. Can use less DNA up to 100ng.

For 1 reaction: 50ul

10x Buffer 5ul 
dNTP's 4ul 
MgCL2 4ul 
Primers 2.5ul each 
TaqPol 0.5ul 
H2O 21.5ul 
DNA 1ug to 100ng in 10ul H2O

Stock solutions:

10x buffer: Perkin-elmer standard PCR buffer w/out MgCl2 should be used. 
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#134 in C209D.

1cycle: 94°C 3min 
30cycles 94°C 45sec 
60°C 45sec 
72°C 2min 
1cycle 4°C forever

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