AptaTaq Fast PCR Master Protocol & Troubleshooting

Product No. ATPAM-RO

Protocol

Procedure

  1. Thaw primer and nucleic acid template solution; mix by vortexing.
  2. Prepare PCR primer solutions (e.g., in a concentration of 10 µM for each primer).
  3. Vortex the AptaTaq Fast PCR Master (vial 1).
  4. Spin down all vials in a microcentrifuge prior to opening to ensure recovery of the entire volume.
  5. To a sterile reaction tube, add the components in the order listed below (for each 20 µl reaction):

Component; Volume; Final Concentration

Water, PCR Grade (vial 2); 12 µl
Forward primer, 10 µM; 1 µl; 0.5 µM
Reverse primer, 10 µM; 1 µl; 0.5 µM
AptaTaq Fast PCR Master, 5x concentrated (vial 1); 4µl; 1x concentrated

Final volume; 18 µl

Note: To prepare fast PCR reaction mixes for more than one reaction, multiply the amount in the column "Volume" by the number of reactions plus sufficient additional reactions.
6. Mix by pipetting.
7. In case of multiple reactions, dispense 18 µl of the reaction mix into individual PCR reaction tubes or wells of a multiwell plate.
8. Add 2 µl nucleic acid template.
9. Mix by pipetting.
10. Place the samples in a thermal block cycler and use the thermal profile below to perform the PCR:

Step; Cycles; Time; Temperature

Initial Denaturation; 1; 30 sec; 95 °C
Denaturation; 25 to 35; 1 sec; 95 °C + Annealing/Elongation; 15 sec; 60 °C
Cooling; unlimited time; 4 °C

Optimization

If the recommended protocol does not fulfill assay requirements, try to optimize the reaction by increasing the annealing/elongation temperature to +63 °C to achieve a higher specificity. In addition, use longer annealing/elongation times for longer PCR products.

Troubleshooting

Issue; Possible Cause; Recommendation

  • No amplification/no product detectable; Error in PCR program; Adjust the PCR program.
  • No amplification/no product detectable; Pipetting errors (e.g., nucleic acid template not added); Repeat experiment - check pipetting steps carefully.
  • No amplification/no product detectable; Amplicon too long; a) Redesign primers to shorten the PCR product, b) Prolong annealing/elongation time.
  • No amplification/no product detectable; Inhibitory effects by impurities of the nucleic acid template; Repeat the isolation of the nucleic acid template.
  • No amplification/no product detectable; Suboptimal primer design; Redesign primers.
  • Amplification products in the negative (no template) control; a) Replace solutions in which a contamination might occur (e.g., water), b) Clean lab environment (e.g., bench), c) Use UNG to prevent carryover contamination.

Materials

     

AptaTaq is a trademark of Roche.

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