Genotyping protocol

protocol

 

1. Tail lysis

*Get the tissue sample from the mouse tail or ear

*Tail lysis buffer(TLB) composition (500ml standard)

  - 1.5M Tris (pH 8.8) 33.3ml

  - 4M NaCl 25ml

  - 0.5M EDTA 5ml

  - 10% SDS 10ml

  - DW

1) Mix Proteinase-K with Tail lysis buffer [proteinase-K: TLB = 1:100]

2) Put 300ul of the mixture in each ep-tube with a tail in it.

3) O/N in 60℃

 

2. DNA extraction

1) Put 700ul of pure EtOH in each ep-tube (final concentration 70%) and mix well

2) Centrifuge for 15min, 12400rpm, in 4℃

3) Remove the supernatant

4) Put 1ml of ice-cold 70% EtOH in each tube and do the inverting

5) Centrifuge for 5min, 12400rpm, in 4℃

6) Remove the supernatant and repeat 4) & 5) steps

7) Remove the supernatant and dry the EtOH 5~10min with the caps open (Room temperature)

8) Put 150ul of Ultra-pure water(nuclease-free water) and incubate it 5min in 60℃

9) Centrifuge for 2min, 12,400rpm, in RT

10) Get the supernatant and store it at -20℃

 

3. PCR(Polymerase Chain Reaction)

1) Make the mixture for PCR (total volume 9ul)

  - Ultra Pure Water (DW) 4ul

  - EconoTaq 2X master mix* 4ul

  - Primer (Fwd 0.5ul + Rev 0.5ul)

    *Can be changed

2) Put the mixture in the PCR tube, 9ul each

3) Put the extracted DNA in the PCR tube, 3ul each

4) Vortex & spin down

5) PCR (refer to the manual from the master mix or lab protocol)

 

4. Electrophoresis

1) Make 1.2% Agarose gel with TAE buffer

2) Put 5ul of Safeview in 1.2% Agarose gel solution(100ml) before it gets hard

3) Load 8ul of sample in each well

4) Run: 135V, 15min in TAE buffer

 

5. Detection