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MOLECULAR BIOLOGY: WORKING WITH DNA

POLYMERASE CHAIN REACTION

BASIC POLYMERASE CHAIN REACTION (PCR) INSTRUCTIONS

Basic Polymerase Chain Reaction (PCR) Instructions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

The most common problem with PCR is the contamination of reactions with foreign DNA. To avoid this, use PCR-dedicated reagents, PCR-dedicated pipettors with plugged tips, and dedicated areas of workspace for PCR reaction preparation and PCR product analysis.

Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. A typical reaction includes:    1X PCR Buffer    5 ng of DNA Template    100 ng Primer A    100 ng Primer B    1X dNTP mix    2.5 Units of Taq Polymerase    Add sterile ddH2O to a final volume of either 50 μl or 100 μl (see Hint #1) 2. Set up PCR reactions in thin-walled PCR tubes on ice. Add Taq Polymerase last or for a "hot start" (see Hint #2). 3. The reaction is divided into 3 repeated cycles. These are typically:    Denaturation at 95°C for 30 seconds    Annealing at between 50°C to 65°C for 1 minute (see Hint #3)    Extension at 72°C for 1 to 2 minutes 4. The number of cycles in each reaction is typically 25 to 40. 5. Denaturation, Annealing, and Extension times need to be determined per reaction. Values for these are dependent on the type of PCR machine used. These values are typically 30 sec for Denaturation and 1 to 2 min each for Annealing and Extension (see Hint #4)

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Nucleotides (10X)    2 mM dGTP 2 mM dCTP 2 mM dATP 2 mM dTTP Magnesium Buffer    25 mM MgCl2 Try concentrations of 1.5, 3.0, 4.5, 6.0, and 10 mM. Autoclave Increasing the Magnesium concentration has the same effect as lowering the annealing temperature. If necessary, Magnesium can be titrated to obtain an optimal concentration. Extra Magnesium can be added to the PCR reaction. If using PCR Buffer, a final Magnesium ion concentration of 1.5 mM will be obtained. PCR Buffer (10X)    500 mM KCl 100 mM Tris-HCl, pH 8.3 15 mM Magnesium Buffer This buffer can also contain 0.1% (w/v) Gelatin Primers    Each primer should be used at approximately 100 ng per reaction. Resuspend oligonucleotides in ddH2O at 100 ng/μl. DNA Template    Use between 1 and 5 ng of cloned DNA or between 40 and 100 ng of genomic DNA per reaction. It is convenient to dilute template stocks to an appropriate concentration (e.g., 5 ng/μl in ddH2O for cloned DNA). Paraffin Oil or Mineral Oil    If the thermocycler has a heated cover, no oil is needed. This prevents the evaporation of the sample. In some thermocycler instruments, either Paraffin or Mineral Oil must be overlayed on the PCR volumes.

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Oligonucleotide Magnesium Chloride Paraffin Oil Tris Mineral Oil Potassium Chloride Gelatin DNA Polymerase, Taq Primer

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. The amount of sterile ddH2O to add depends on the total amount of product desired. 2. A Denaturation step is included prior to the start of the reaction cycles. This denaturation is usually a 4 min step at 95°C. In a "hot start" PCR reaction, the Taq Polymerase is added to the reaction tubes immediately after this initial Denaturation step. This is to prevent formation of inappropriate products or excessive primer dimers that might occur before the first denaturing, annealing, and extension cycle. 3. The appropriate Annealing temperature must be determined for each pair of primers and template. Reactions should be carried out at a number of different annealing temperatures to determine an optimum value. Start with an annealing temperature that is 5°C below the calculated melting temperature of the primers. 4. Longer PCR products (greater than 1 kb) need longer extension times.