PCR Tips and Tricks

General Guidelines

• Reagent concentrations - "less is usually better"  (more specific)

  • primers: final concentration 0.1-1.0 uM

  • MgCl₂:   final concentration 1.0-4.0 mM (depends on Taq used)

  • dNTPs:  final concentration 0.2 mM each dNTP (depends on Taq used)

    • note: sensitive to repeated freeze/thaws

• Vortex or finger-flick reagents to mix well before use

• Primer design very important

  • the higher the annealing temperature the better (T_ANN > 50°C)

  • primer pairs should have melting temps within 5°C of each other

• Annealing temperature and step times are important

• Titrations are a good idea

  • most commonly temperature and MgCl₂

  • reactions often borderline resulting in inconsistent amplifications

  • use Eppendorf Mastercycler Gradient for titrations

  • titration on Mastercycler Gradient

  • sample titration gel

• Hot starts improve reaction efficiency (fewer primer-dimers)

  • manual: add Taq to tubes in thermalcycler at 94^oC (or MgCl₂ or dNTPs)

  • TaqStart Antibody (Clontech)

  • Faststart Taq (Roche)

  • hotstart comparison gel

• Always check program on thermalcycler

• Run negative control(s) to check for contamination

• Make a flow chart of what tried and in what order

• Run a positive control (a sample known to amplify well)

• Always run a ladder on gel (will indicate whether failed PCR or failed detection system)

• Additives for fragments that are very long, G-C rich or prone to secondary structure

  • glycerol, formamide, NMP - lower denaturing and annealing temperatures by a few degrees

  • DMSO decreases incidence of secondary structure

• For more tips see:   http://www.biowire.com/nucleus/nucleus_1_1.jsp

Troubleshooting

• Weak or no amplification:

  • increase # cycles

  • increase time at step(s)

  • add final extension of 10 min

  • increase quantity of template

  • increase Taq concentration by 2X

  • try different Taq (e.g. Faststart)

  • increase MgCl₂, primers, dNTPs

  • reamplify PCR product from previous reaction

    • PCR reamplification protocol

• Non-targeted bands:

  • high weight bands: decrease Taq concentration

  • low weight bands: decrease MgCl₂ concentration

  • decrease # cycles

  • raise annealing temperature

  • decrease primer concentration

  • decrease dNTPs

• Possible inhibition

  • dilute template 5:1 with 10mM Tris-HCl (pH 8.0) or ddH₂O

  • add BSA - final concentration 1X

  • see http://www.biowire.com/nucleus/nucleus_1_1.jsp

• +A problem

  • add final extension of 45 min at 60°C

• Smears above band

  • decrease # cycles

• Carpet

  • decrease concentration of reagent(s)