NOTICE: You are viewing a page of the openwetware wiki. Our "dewikify" feature makes a wiki page appear as a normal web page. In April 2017, this feature will GO AWAY and this URL will redirect to the source URL on our wiki. We're sorry for the inconvenience.

Back to Eccles Lab > DGG Protocols

Contents

Mouse Pax2-1Neu PCR for WAVE DHPLC genotyping

For detecting Pax2-1Neu heterozygous mutant mice using heteroduplex analysis via DHPLC (denaturing high performance liquid chromatography) on the Transgenomic WAVE machine (Robertson Lab). Not all Taq enzymes and associated buffers are able to be used on the WAVE – AmpliTaq Gold, Roche standard and FastStart Taq, and Stratagene's AccuType Taq, to name a few, have been authorised by Transgenomic for use on the WAVE (use of non-compliant Taq will void HPLC cartridge warranty). The wild type allele gives a single peak, whereas the mutant gives an unambiguous double peak. Read appropriate bits of the Transgenomic manual before proceeding.

Materials

Taq

PCR primers

Reaction conditions

Set up the following on ice:

Reagents 50 uL 25 uL
DNA 10-50 ng 10-50 ng
Primer 2292 (10 uM) 0.5 uL 0.25 uL (200 uM final)
Primer 2293 (10 uM) 0.5 uL 0.25 uL (200 uM final)
dNTP's (10 mM) 1 uL 0.5 uL (200 nM final)
10 x plus Mg2+ buffer 5 uL 2.5 uL (1.5 mM final)
Taq 1 U 0.5 U
Water to 50 uL to 25 uL
Total 50 uL25 uL

Thermal cycling

  1. 94°C, 2:00 min.
  2. 94°C, 30 sec.
  3. 60°C, 30 sec.
  4. 72°C, 30 sec.
  5. To step 2 for 29 cycles (30 cycles total).
  6. 72°C, 7:00 min.
  7. 10°C hold.

Heteroduplexing

After thermal cycling, the resulting amplicons need to be heteroduplexed to allow detection on the WAVE. This is achieved by heating and then slowly cooling to (i) denature the DNA, and (ii) allow single strands to re-anneal to result in (iii) a mixture of homoduplexes (wt:wt) and heteroduplexes (wt:mut). The heteroduplexing step can be added to the end of the normal cycling profile, or done in a separate step any time prior to the reactions being loaded onto the WAVE. The heteroduplexes will only form if the Pax21Neu insertion mutation is present , and will result in two peaks on the WAVE (the first peak resulting from melting of heteroduplex and the second homoduplex). There are heteroduplexing programs on both the MJ Diad (Robertson Lab), and the PTC-100 and Biometra (Eccles lab) consisting of:

Amplicon length

167 (wt), 168 (mutant). Check 6 uL (50 uL PCR) or 3uL (25 uL PCR) product on 1-2% agarose gel.

A. Jeffs, June 2003.