TRFLP Community Analysis of a PCR product

• one or more four-bp cutting restruction enzymes (e.g. Alu, MnlI, RsaI, Sau3A)
• pellet pait
• prepare 8 μl pellet paint as follows:

 6 μl glycogen
 2 μl AB Loading Dye (no salt)

• 3 M NaAcetate pH 5.2
• dilute this 1:4
• 95% ethanol (use the molecular grade ethanol)
• 70% ethanol

• quantify your DNA by running a gel of your PCR product and comparing band intensity to those of duplicate lanes of lambda-HindIII standard
• dilute PCR product to 5 ng μl-1
• Digest your PCR product in the following reaction mix:

 3 μl DNA (15 ng)
 2 U Restriction Enzyme (for today use 1 μl)
 2 μl 10 X Buffer
 13 μl DI water

• Digest for at least 4 hours at 37 deg. C
• After digestion add:

 1.9 μl NaAcetate (1:4)
 0.4 μl Pellet Paint

• vortex well (!)
• add 37 μl 95% ethanol
• vortex well (!)
• place in -20 dec C freezer for 20 min
• spin at 13,000 rpm for 10 min
• remove supernatant
• add 100 μl 70 % ethanol
• spin at 13,000 rpm for 10 min
• remove supernatant
• dry in speedyvac for at least 30 min
• Add 20 μl de-ionized formamide and 0.3 μl ROX size standard
• denature at 95 deg C for 5 min
• place on ice for 5 min
• run sample on sequencer