PCR基本实验方法（四）

Labelling PCR Products with Digoxigenin

PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790). This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.

Make a DIG-dNTP mix for PCR as follows:

DIG NUCLEOTIDE MIX CONCENTRATIONS

Dig-11-dUTP 350 uM 　　dTTP 650 uM 　　dATP 1 mM 　　dCTP 1 mM 　　dGTP 1 mM

For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above. The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically. Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured. Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature.

Cleaning PCR Products

Getting rid of mineral oil: simply add 50ul of chloroform to the reaction vial, vortex and centrifuge briefly, and remove the "hanging droplet" of AQUEOUS solution with a micopipette.

Getting rid of wax or Vaseline: simply "spear" wax gem and remove; do as for oil or bottom-puncture tube and blow out aqueous ｄｒｏｐ for Vaseline. Cleaning-up DNA: 3 options

a protocol which gives DNA that is clean enough for sequencing is the following: increase volume to 100ul with water, add 10M ammonium acetate soln. to 0.2M final concentration (1/5th volume), add equal volume of isopropanol (propan-2-ol), leave on bench 5 min, centrifuge 20 min at 15 000 rpm, remove liquid using pipette, resuspend in 100ul water or TE, repeat precipitation. Simply do a phenol-CHCl3 extraction (add 20ul phenol to aqueous phase, vortex, add 50ul CHCl3, vortex, centrifuge, remove UPPER aqueous phase, repeat CHCl3 extraction). Make aqueous phase up to 400ul, and spin through Millipore Ultrafree-MC NMWL 30 000 cartridges (at 6000 rpm in microcentrifuge), wash through with 2x400ul water, collect +/-20ul sample: this is pure enough for sequencing.

NOTE:

Product is clean enough for restriction digest immediately after reaction; however, phenol-chloroform clean-up is recommended as a minimum.

Sequencing PCR Products:

This is best done using ssDNA generated by asymmetric PCR, and the "limiting" primer for sequencing. However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. (1990) (NAR 18: 1309). CLEAN DNA is resuspended in sequencing buffer containing 0.5% Nonidet P-40 and 0.5% Tween-20 and sequencing primer, denatured by heating to 95oC for 5 min, snap-cooled on wet ice, and sequenced by the "close-to-primer" protocol (eg: dilute extension mixes).