Alkami Quick Guide™ for PCR

•Tris-HCl: 10 mM - 67 mM, pH 8.2 - 9.0

•KCl: 25 mM - 50 mM

•MgCl2: 1.5 - 5.0 mM

•Gelatin: 0.01% - 0.1% (w/v)

•Non-ionic detergents

•Tween 20: 0.05% (v/v)

•Triton-X-100: 0.01% (v/v)

•Tetramethyl ammonium bromide

•TMANO (trimethylamine N-oxide)

•Betaine

How to Reduce Contamination

Possible Sources of Contamination: Components

Reagents

•Purified restriction fragment of target sequence

•Plasmid DNA that contains target sequence

•Post PCR contamination from the handling of PCR products.

Possible Sources of Contamination: Conditions

•Biological samples (e.g. patients, animal, etc.)

•Sample collection methods

•Lab Staff

•Lab environment

•Liquid nitrogen/ice

•Tissue homogenizer

•Gloves

•Pipetts/pipette tips

•Reaction tubes/glassware

•Recombinant or biological products (e.g. gelatin, bovine serum albumin, restriction enzymes)

•Hood or fume-cupboard filters

www.Alkami.org

•Centrifuges/centrifuge tubes

•Thermal cycler, heating blocks, water baths

•UV transilluminator

•Electrophoresis apparatus

•Dot-blot apparatus

•Razor and/or microtome blades

•Microcentrifuges

•Concentrators and vacuum bottles

•Gel apparatus or UV light box

•Dry ice/ethanol or water baths

Techniques Used to Prevent Contamination

DNA Polymerase Contaminants (specifically Taq)

•Aliquot your reagents. Reagents stored as aliquots minimize repeated samplings. Contamination of reagents with target DNA can be checked by doing your PCR in the absence of exogenously added DNA.

•Add non-sample components to the reaction mixture before adding your sample DNA and cap each tube before proceeding to the next sample.

•Derive singleand double-stranded DNA with chemical adducts, such as isopsoralen.

•For prevention of contamination by dUDP-labeled PCR products, the use of uracil DNA-glycosylase (UNG) and deoxyuridine triphosphate (dTTP) substituted for thymidine triphosphate (dTTP).

Conditions

•Physical separation of the individual parts of the PCR into sample preparation, pre-PCR, and post-PCR locations.