1. Quantitate tail DNA.
2. Prepare 20 µl aliquots of tail DNA diluted to 200 ng/microliter in autoclaved water.
3. Heat DNA for 1 minute in a dry bath set for 95 degrees C. Spin down tubes place on ice.
4. Prepare PCR cocktail.
|Reagents||for 1 reaction||for 20 reactions
|autoclaved water ||14.0 microliters ||280.0 microliters
|10X PCR Buffer ||2.5 microliters ||50.0 microliters
|Nucleotides (10 millimolar) ||4.0 microliters ||80.0 microliters
|Primer A (10pmol/microliter) ||1.25 microliters ||25.0 microliters
|Primer B (10pmol/microliter) ||1.25 microliters ||25.0 microliters
|Taq Polymerase (0.3125 U/microliter) ||1.0 microliters ||20.0 microliters
|Total Volume ||24. microliters ||480.0 microliters
5. Aliquot 24.0 microliter of cocktail into PCR tubes.
6. Add 1.0 microliter of heat treated DNA to PCR tube (if tail DNA is less than 200 ng/microliter then add 200 ng of DNA in a volume up to 3 microliters).
7. Final volume of PCR reaction is 25.0 microliters.
8. Overlay with mineral oil and place into thermal cycler.
9. Amplify 30 to 35 cycles.
10. Analyze on appropriate ethidium bromide containing agarose gel. We analyze up to 60 samples on a 20 X 20 cm 1.5% agarose gel in TBE.
- Prepare a cocktail with an extra 10% to account for pipetting errors.
- Remember to include a negative control of water only and a positive control of known transgenic mouse DNA or linearized plasmid DNA diluted in mouse tail DNA to the one copy level.
- Remember to include an internal positive control to ensure that the DNA sample is “amplifiable.” For this purpose we use primers for mouse beta globin. Primers that amplify any single copy mouse gene may also be used. For example, amplification of beta globin indicates that the DNA is clean and “amplifiable.” This will prevent false negative results and positive animals will not be accidentally discarded.
- Internal positive control primers may be combined with transgene specific primers if the two primer sets do not interfere with each other.
- Use primer picking software. We have had very good results with Primer3.