A Quick, No Frills Approach to Mouse Genotyping

Materials and Reagents

1. NaOH (NaOH pellets)
2. Taq DNA Polymerase with ThermoPol buffer (New England Biolabs, catalog number:  M0267X *, M0267L , or M0267S )
   * Note: At 4,000U, ~800 μl of Taq serves several thousand PCR reactions. Buffer becomes a limiting reagent. ThermoPol Buffer recipe is available at NEB website. This buffer can be ordered separately from NEB (New England Biolabs, catalog number:  B9004S )
3. Primers, recommend to be 18-21 bp in length, have a melting temperature above 56 °C and around 58 °C, and produce amplicons of 150-600 bp.
4. DNA loading buffer
   Recommend Orange G (Sigma-Aldrich, catalog number:  O3756 ) instead of bromophenol blue for loading dye
5. DNA ladder range for 100-800 bp range
   Recommend 1 kb Plus DNA ladder (Life Technologies, catolog number: 10787-018 )
6. Agarose
7. EtBr (Sigma-Aldrich, catalog number:  E8751 ) or SYBR Safe DNA Gel Stain (Life Technologies, catalog number: S33102 )
8. NaOAc
9. EtOH
10. Phenol/Chloroform/Isoamyl alcohol (25: 24: 1) (Do not use acid phenol)
11. 50 mM NaOH in dH₂O (see Recipes)
    
12. 10 mM dNTP Mix  (see Recipes)
    
13. 1x TAE buffer (see Recipes)
    
14. dNTP set 100 mM each A, C, G, T (GE Life Sciences, catalog number: 28406552 ) (see Recipes)
    
15. 0.3 M NaOAc in ddH₂O (see Recipes)
    

Equipment

1. PCR Thermal cycler (96 well capacity preferred)
2. Centrifuges (mini for PCR tubes and microcentrifuge for 1.5 ml tubes)
3. Liberty 1 buffer-less high speed gel system (Neuvitro, 6Mgel - SYS-LBT1)
4. Liberty 1, 12-channel pipette compatible 13 teeth combs (Neuvitro, 6Mgel - CMS-1315)
5. Multichannel Pipette 2-20 μl
6. Repeat Pipettor 10-125 μl
7. PCR tube with cap, 8 or 12 PCR strip tubes, or 96-well PCR plate
   

Procedure