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Thursday, May 30, 2013

12 Things to rememeber while Setting up a PCR Reaction


12 Things to rememeber while Setting up a PCR Reaction
This is a guest post by one of the blog readers.

setting up a PCR reaction
  • Consumables for setting up a PCR reaction such as PCR Tubes, Pipettes, PCR Racks should be cleaned with Hypo and then expose to UV for atleast 20 minutes.
  • Before Setting up a PCR reaction Wash Hands,Wear Gloves.
  • Always use Aerosol barrier tips (ART - Aerosol Resistant pipette Tips) for setting up PCR reaction, changing tips for each step.
  • Use PCR grade water for diluting the stocks and for setting up a PCR Reaction.
  • Use separate rooms for performing Mastermix preparation, Sample addition & Post PCR.
  • Always use fresh aliquot of Reagents.
  • Reagents for PCR should be thawed completely,vortex it and centrifuge before use.
  • Never enter Post PCR room before setting up your Reaction.
  • Accurate Pipette handling is must - Use calibrated pipettes.
  • Start the PCR Reaction immediately after setting up the reaction this will help in avoiding Primer dimer formation.
  • Positive and Negative controls should be kept in each PCR reaction without that PCR reaction is not considered as valid.
  • Always perform PCR in Duplicates or in Triplicates.
  • Make Sure you have cross checked the calculations before proceeding with the reaction.
Contamination in PCR lab is a Disaster....!!! Handle PCR Products (amplicon) Carefully.