Download Troubleshooting Guide for End-Point PCR

Troubleshooting Guide for End-Point PCR
Problem
No product or low yield
Non specific products
Product in negative control
Possible Solution
Make sure you added all reaction components (e.g.
template DNA, primers, buffers, MgCl2 etc.).
Make sure there are no inhibitors in DNA sample (e.g.
heparin)
Double check the sensitivity and compatibility of your
primers.
Verify that there are no primer-dimers.
Try increasing MgCl2 concentration.
Increase primer amount.
Increase template amount.
Verify template purity and integrity.
Change the dNTP solution.
®
If using HOT FIREPol , make sure you wait for the
activation time (12 to 15 minutes initial heating).
Gradually increase extension time.
Gradually decrease annealing temperature.
Add enhancing agents e.g. DMSO, PEG, BSA,
Solution S.
Add increments of MgCl2.
Check that the wells on the gel are loaded correctly
and that the loading buffers and ethidium bromide are
added
If using normal PCR, try using Hot Start instead.
Lower MgCl2 concentration.
Try all the buffers provided.
Lower template concentration.
Lower annealing temperature gradually.
Decrease annealing time.
Lower primer concentration.
Increase extension time.
Make sure you do not have contamination from
impure template or reagents.
Always use filter tips. Wear gloves. Avoid leaving
tubes lids open to avoid aerosol contamination.
Solis BioDyne
Riia 185a, 51014 Tartu, Estonia, tel: +372 740 9960, fax: +372 740 2079, e-mail: [email protected], www.sbd.ee