Protocol for Routine PCR Directly from Whole Blood or Dried Blood Spots (DBS) using Q5^® Blood Direct 2X Master Mix (NEB #M0500)

Please note that protocols with Q5 Blood Direct 2X Master Mix may differ from protocols with other polymerases or versions of Q5. Conditions recommended below should be used for optimal performance.

Protocol for Routine PCR from Whole Blood

Reaction setup:

Q5 Blood Direct 2X Master Mix is inhibited at room temperature, allowing flexible reaction setup (room temperature or ice). All components should be mixed prior to use.

1. In PCR tubes or 96-well plate, combine the following except whole blood:

    

   Component	20 µl Reaction	50 µl Reaction	Final Concentration
   Q5 Blood Direct 2X Master Mix	10 µl	25 µl	1X
   10 µM Forward Primer	1 µl	2.5 µl	0.5 µM
   10 µM Reverse Primer	1 µl	2.5 µl	0.5 µM
   Whole Blood (Human)	Variable*	Variable**	5-10% (≤40%)
   Nuclease-Free Water	to 20 µl	to 50 µl

   *5-10% in 20 µL reaction
   **≤40% in 50 µL reaction
   
   

2. Gently mix components and collect the liquid at the bottom of the tube by a quick spin.
   
   
3. Add blood last, allowing it to sink to the bottom of the tube; do not mix after adding.
   
   
4. Transfer PCR tubes or plate to a PCR machine and begin thermocycling.

Thermocycling Conditions for Routine PCR Directly from Blood:

*Use of the NEB Tm Calculator is highly recommended.
**For amplicons ≥1 kb, extension times of 30-90 seconds/kb and 35-40 cycles are recommended.

Protocol for Routine PCR from Dried Blood Spots

Reaction setup:

1. In PCR tubes or 96-well plate on ice or at room temperature, combine the following except for the DBS punch:

    

   Component	20 µl Reaction	50 µl Reaction	Final Concentration
   Q5 Blood Direct 2X Master Mix	10 µl	25 µl	1X
   10 µM Forward Primer	1 µl	2.5 µl	0.5 µM
   10 µM Reverse Primer	1 µl	2.5 µl	0.5 µM
   Punch from Dried Blood Spot	1 x 0.5 mm	1 x 1 mm
   Nuclease-Free Water	8 µl	20 µl

2. Gently mix components and collect the liquid at the bottom of the tube by a quick spin.
   
   
3. Add DBS punch last; do not mix after adding.
   
   
4. Transfer PCR tubes or plate to a PCR machine and begin thermocycling.

Thermocycling Conditions for Routine PCR Directly from Blood Punches:

*Use of the NEB Tm Calculator is highly recommended.
**For amplicons ≥1 kb, extension times of 30-90 seconds/kb and 35-40 cycles are recommended.

General Guidelines:

1. Template:

DNA does not need to be purified from blood. Whole human blood preserved with K₂ EDTA, Na EDTA, Na citrate and Na heparin are all suitable for PCR with Q5 Blood Direct 2X Master Mix. PCR from non-human blood may require additional optimization. Although up to 40% human blood (v/v) may be used, best results are usually achieved with 5-10% blood (e.g., 1-2 µL blood in a 20 µL reaction). For higher blood volumes (≥20%), a 50 µL reaction volume is recommended due to difficulties in recovery of the aqueous phase from blood cell debris that remains after PCR.

For amplification from dried blood spots stored on Whatman^® 903 and FTA papers, we recommend starting with a single 0.5 mm punch in a 20 µL reaction or a single 1 mm punch in a 50 µL reaction.

DBS Punch treatment: 

In general, Whatman^® 903 and FTA papers spotted with blood preserved with EDTA, citrate or heparin can be used without pre-treatment. However, if more punches or larger punches are added, they can be pre-treated to reduce possible PCR inhibition. To pre-treat, add each DBS punch to a separate PCR tube containing 50 µL nuclease-free water and heat at 50°C for 5 minutes. Remove punches and proceed with step 1 of the DBS Punch protocol. Alternatively, liquid may be removed by pipette after heat treatment and cocktail can be added directly to PCR tubes containing the washed punches.

Primers:

Oligonucleotide 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 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.

Mg++:

The Q5 Blood Direct Master Mix contains 2.0 mM Mg++ when used at a 1X concentration. This is optimal for most PCR products generated with this master mix. For particularly difficult targets, increasing the Mg++ concentration by an additional 1-4 mM may increase the yield.

Deoxynucleotides:

The final concentration of dNTPs is 200 µM of each deoxynucleotide in the 1X Q5 Blood Direct Master Mix. The Q5 Hot Start High-Fidelity DNA Polymerase in the Q5 Blood Direct 2X Master Mix cannot incorporate dUTP and is not recommended for use with uracil-containing primers.

Denaturation:
Q5 Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.

An initial denaturation step of 3 minutes at 98°C is recommended to thoroughly lyse the blood cells and release/denature the DNA.

During thermocycling, denaturation should be kept to a minimum. Typically, a 1 second denaturation at 98°C is sufficient.


Annealing:

Optimal annealing temperatures for Q5 Blood Direct 2X Master Mix tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically, a 5-second annealing is recommended. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.

Extension:

The recommended extension temperature is 72°C. Extension times are generally 15-30 seconds for amplicons up to 1 kb. Extension time can be increased to 30-90 seconds/kb for amplicons ≥1 kb.

A final extension of 2 minutes at 72°C is recommended.  

Cycle number:

Generally, 30-40 cycles yield sufficient product. For difficult or long amplicons (≥1 kb), at least 35 cycles are recommended.