PCR Protocol for Hemo KlenTaq^® (M0332)

Overview

The following guidelines are provided to ensure successful PCR using New England Biolabs' Hemo KlenTaq DNA Polymerase.

Protocol

Before use, thoroughly mix 5X Hemo KlenTaq Reaction Buffer by inversion.

1. Add to a thin-walled PCR tube on ice:
   

   COMPONENT	25 µl	50 µl	FINAL CONCENTRATION
   5X Hemo KlenTaq Reaction Buffer	5 µl	10 µl	1X
   10 mM dNTP	0.5 µl	1 µl	0.2 mM
   Hemo KlenTaq	2 µl	4 µl
   10 µM Forward Primer	0.75 µl	1.5 µl	0.3 µM (0.05-1 µM)
   10 µM Reverse Primer	0.75 µl	1.5 µl	0.3 µM (0.05-1 µM)
   Whole Blood	up to 2.5 µl*	up to 10 µl**
   Nuclease-free Water	to 25 µl	50 µl

   * ≤=10% Recommended in 25 µl reaction volume.
   ** ≤=20% Recommended in 50 µl reaction volume.
2. Before adding blood, thoroughly mix components by pipetting up and down.
3. Add blood last and let sink to the bottom of the tube.
4. Transfer PCR tubes to a PCR machine with a preheated block at 95°C and start cycling.
5. For reactions containing >10% blood, a 50 µl reaction volume is recommended.
   
   DNA Template: Whole blood samples treated with sodium heparin, sodium EDTA, potassium EDTA, or sodium citrate are recommended. Although up to 30% blood (v/v) can be used in the PCR reactions, 5%–10% is recommended. High concentrations of blood are not recommended due to difficulties in recovery of the aqueous supernatant from blood cell debris that remains after the reaction. Dry blood stored on Guthrie cards or 903 cards (Whatman, NJ) can be used directly by adding 1 mm punch disc to a 25 μl PCR reaction. If blood is stored on FTA paper (Whatman, NJ), incubating the 1 mm punch disc first in 50 μl water at 50°C for 5 minutes is recommended. Discard the 50 μl water and use the disc directly in a 25–50 μl PCR reaction. For GC-rich targets, up to 10% DMSO can be included (1). 
   
   Primers: Primers are generally 20–30 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as PrimerSelect™ (DNAStar Inc, Madison, WI) and Primer3 (https://bioinfo.ut.ee/primer3) can be used to design or analyze primers.
   The final concentration of each primer in a typical PCR reaction is 0.05–1 μM, ideally 0.3 μM. 
   
   Deoxynucleotides: The concentration of dNTPs is typically 200 µM of each deoxynucleotide. 
   
   Starting Reactions: Hemo KlenTaq has increased cold sensitivity, conferring some hot start character on the enzyme (2). Nonspecific priming can be minimized by assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).
   
   Denaturation Temperature and Duration: An initial 3 minute denaturation step at 95°C is recommended prior to PCR cycling to fully lyse the blood cells and release/denature the DNA. 
   
   Annealing Temperature and Duration: The annealing step is typically 30 seconds to 1 minute. The annealing temperature can be optimized by doing a temperature gradient PCR starting 5°C below the calculated melting temperature (T_m). We recommend using NEB's Tm Calculator to determine appropriate annealing temperature for PCR.
   
   Extension Time: The recommended extension temperature is 68°C. A final extension of 10 minutes at 68°C is recommended. The extension rate is generally 2 minutes per kb.
   
   Cycling Conditions: Generally, 30–40 cycles give optimal amplification.
   
   

   STEP	TEMP	TIME
   Initial Denaturation	95°C	3 minutes
   30-40 Cycles (Typically 35)	95°C 50-68°C 68°C	20 seconds 30 seconds 2 minutes per kb
   Final Extension	68°C	10 minutes
   Hold	4-10°C

References:
1.  Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques . 15, 372-374.
2.  Kermekchiev, M.B., et al. (2009). Nucleic Acids Res. 37, e40.