PCR Troubleshooting

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)