Protocols

DNA Isolation from Tails

1. NaOH extraction (quick "dirty" DNA preparation). Reference: Truett GE et al. 2000. Biotechniques 29(1):52-54
2. Cut 2 mm of tail and place into an Eppendorf tube or 96-well plate
3. Add 75ul  25 mM NaOH / 0.2 mM EDTA (Alkaline Lysis Reagent)
4. Place in thermocycler or heat block at 95ºC for 1 hour, then reduce the temperature to 15°C until ready to proceed to the next step
5. Add 7 5ul of 40 mM Tris HCl (pH 5.5)
6. Centrifuge at 4000 rpm for 3 minutes
7. Take an aliquot for PCR (use 2 ul undiluted, or 2 ul of a 1:100 dilution/reaction)

Alkaline Lysis Reagent

To 25 ml water, add:

• 62.5 µl of 10 N NaOH (final concentration is 25 mM.)
• 10.0 µl of 0.5 M disodium EDTA (final concentration is 0.2 mM, pH should be about 12 but should not have to be adjusted.)

Make fresh every one to two months. Keep solution at room temperature.

Neutralization Reagent

Make 1M Tris: 121.14 g Tris base, pH to 7.0 with HCl à 1 L final volume; autoclave

Add 4 ml Tris to ~80 ml water. pH to 5.5 à Make up to 100 ml final volume (40 mM final [ ])

Genotyping

1. Make up reaction mix

Component	Volume	X ____ Samples
5x Green GoTaq Flexi Buffer	4 ul
MgCl2 (25 mM)	1.6 ul
Nucleotide Mix (10 mM)	0.4 ul
GoTaq DNA Pol. (5U/ ml)	0.1 ul
PCR Grade H2O	3.9 ul
Total Volume	10 ul
Transfer Volume	10 ul

 

 

2. Make up assay mixture

Component	Volume	Final [ ]
Tail DNA	1 ul	<0.25 ug
Primer 1 (10 mM)	1 ul	0.5 uM
Primer 2 (10 mM)	1 ul	0.5 uM
PCR Grade H2O	7 ul
Total Volume	10 ul

 

3. Add master mix (10 ul) to each assay (10 ul) for a total volume of 20 ul

4. Run appropriate PCR program

5. Prepare agarose gels

• 1% gel: 200 ml 1x TAE Buffer + 2 g Agarose (+ 5 ul SybrSafe)
• 2% gel: 200 ml 1x TAE Buffer + 4 g Agarose (+ 5 ul SybrSafe)

6) Load 20 ul of the PCR reactions directly into agarose gels.

• Include 10 ul of 100 bp ladder for each row
• Include (+) and (-) controls when possible

7) Run gels at a constant 120 volts. Will take approximately 30 to 40 minutes for large gels. 

8) Photograph using AlphaImager Gel Imager