REDExtract-N-Amp™ Blood PCR Kits Protocol

Catalog Numbers: XNABS, XNAB, XNABE, XNABR, XNABRE, and P8240

Product Description

The REDExtract-N-Amp Blood PCR Kits contain all of the reagents required to rapidly extract and amplify human genomic DNA from whole blood, whole blood dried on a blood card, and cultured mammalian cells. Briefly, DNA is released by incubating the sample with the Lysis Solution at room temperature for 5 minutes for whole blood, at 55 °C for 15 minutes for blood cards, or at 75 °C for 5 to 10 minutes for cell monolayers. After adding the Neutralization Solution, the extract is ready for PCR.

An aliquot of the neutralized extract is then combined with the REDExtract-N-Amp Blood PCR ReadyMix™ and user-provided PCR primers to amplify the target DNA. The REDExtract-N-Amp Blood PCR ReadyMix is a 2x reaction mixture containing buffer, salts, dNTPs, and Taq polymerase. It also contains the JumpStart™ Taq antibody for hot start PCR to enhance specificity and the REDTaq® dye to allow direct loading of the PCR product onto an agarose gel.

 

Reagents Provided Catalog
Number
XNABS
10 preps,
10 PCRs
XNAB
100 preps,
100 PCRs
XNABE
100 preps,
500 PCRs
XNABR
1,000 preps,
1,000 PCRs
XNABRE
1,000 preps,
5,000 PCRs
Lysis Solution for Blood L3289 0.3 ml 2.5 ml 2.5 ml 25 ml 25 ml
Neutralization Solution for Blood N9784 2 x 1.5 ml 25 ml 25 ml 250 ml 250 ml
REDExtract-N-Amp Blood PCR ReadyMix.
This is a 2
X PCR reaction mix containing
buffer, salts, dNTPs,
Taq
polymerase, REDTaq
dye, and JumpStart™Taq antibody.
P8240 0.15 ml 1.2 ml 5 x 1.2 ml 12 ml 5 x 12 ml

Storage

The REDExtract-N-Amp Blood PCR Kits can be stored at 2-8 °C on a short-term basis up to 3 weeks. For long-term storage greater than 3 weeks, -20 °C is recommended. Do not store in a "frost-free" freezer.

Reagents and equipment required but not provided

  • Microcentrifuge tubes or multiwell plate for extractions (200 µL minimal volume)
  • Punch and cards for dried blood
  • Incubator or oven for blood cards (55 °C) or monolayer cells (75 °C)
  • Tubes or plate for PCR
  • Thermal cycler
  • PCR primers
  • Water, PCR reagent, Catalog Number W1754

Precautions and Disclaimer

The REDExtract-N-Amp Blood PCR Kits are for R&D use only, not for drug, household, or other uses. The Lysis Solution is caustic. Avoid contact with skin. Wear gloves, safety glasses, and suitable protective clothing when handling this or any other reagent provided with the kit. Consult the SDS for information regarding hazards and safe handling practices.

Procedure

All steps are carried out at room temperature unless otherwise noted.

A. DNA extraction from Whole Blood
1a.   Collect blood into tubes containing EDTA, sodium citrate, or sodium heparin. The best results may be obtained
        with EDTA or sodium citrate. Mix thoroughly by inversion or rocking.
        Note: For non-human sources, collect blood into tripotassium EDTA, Catalog Number E0270, at a final
        concentration of 5 mM to prevent coagulation.

2a.    Place 20 µL of the Lysis Solution for Blood into a microcentrifuge tube or well of a multiwell plate for each
         extraction.

3a.    Add 10 µL of blood. Mix thoroughly by vortexing or pipetting.

4a.    Incubate at room temperature for 5 minutes.

5a.    Add 180 µL of the Neutralization Solution for Blood. Mix thoroughly by vortexing or pipetting.

6a.    Store the neutralized blood extract at 4 °C or use 2 µL immediately in PCR. Continue with step 7.
         Note: DNA is stable in the extract for at least 6 months at 4 °C.

B. DNA extraction from Blood Cards
1b.    Collect the blood sample on to a collection card, such as Catalog Number Z719838. Allow to dry completely.

2b.    Punch a disk (preferably 1/8 inch or 3 mm) from the blood card and place into a microcentrifuge tube. Make
         sure that the punch contains as much of the blood-stained area as possible.

3b.    Pipette 20 µL of the Lysis Solution for Blood onto the blood card punch. Samples can be spun in a
         microcentrifuge for a few seconds to force the solution into the punch.

4b.    Incubate at 55 °C for 15 minutes.

5b.    Add 180 µL of the Neutralization Solution for Blood. Mix thoroughly by vortexing or pipetting.

6b.    Store the neutralized blood extract at 4 °C or use 2 µL immediately in PCR. Continue with step 7.
         Note: DNA is stable in the extract for at least 6 months at 4 °C.

C. DNA Extraction from Cultured Mammalian Cells
1c.    Grow monolayer cells in a multiwell plate until 90 to 95% confluent.

2c.    Aspirate the medium from the wells using a pipette tip connected to a vacuum system. The medium must be
        removed completely.

3c.    Add 20 µL of the Lysis Solution for Blood to each of the wells.
        Note: It is preferred at this point to seal the plate with AlumaSeal™ II, Catalog Number A2350, to prevent loss
        by evaporation during incubation in step 4c. The Alumaseal can be pierced with a pipette tip to add the
        Neutralization Solution for Blood in step 5c. A new layer of AlumaSeal can be placed over the original layer to
        reseal the plate for storage.

4c.    Incubate the plate at 75 °C for 5 to 10 minutes (for a 24 well plate, 5 minutes is recommended to avoid
         overdrying the samples).

5c.    Add 180 µL of the Neutralization Solution for Blood to each of the wells. Mix the samples by pipetting up
        and down.

6c.    Store the neutralized cell extract at 4 °C or use 2 µL immediately in PCR. Continue with step 7.
         Note: DNA is stable in the extract for at least 6 months at 4 °C.

PCR amplification

The REDExtract-N-Amp Blood PCR ReadyMix contains the JumpStart Taq antibody for specific hot start amplification.  Therefore, PCR reactions can be assembled at room temperature without premature Taq DNA polymerase activity.

Typical final primer concentrations are approximately 0.4 µM each. The optimal primer concentration and cycling parameters will depend on the system used.

7.    Add the following reagents to a thin-walled PCR microcentrifuge tube or plate:

 

Reagent Volume
Water, PCR Reagent x µL
REDExtract-N-Amp Blood
PCR ReadyMix
10 µL
Forward primer y µL
Reverse primer y µL
Neutralized blood extract 2 µL
Total volume 20 µL

Note: The neutralized blood extract may inhibit PCR amplification of products larger than 2 kb. Neutralization Solution B, Catalog Number N3910, can be used to overcome this inhibition and allows successful amplification of longer PCR products. Add 1 µL of Neutralization Solution B to each reaction. Neutralization Solution B is not part of this kit and must be purchased separately.

8.    Mix gently.

9.    For thermal cyclers without a heated lid, add 20 µL of mineral oil on top of the mixture in each tube to
       prevent evaporation.

10.  Perform thermal cycling. The amplification parameters should be optimized for individual primers, template, and
       thermal cycler (see References for guidance).

Common cycling parameters:

Step Temp. Time Cycles
Initial
Denaturation
94-96 °C 3 minutes 1
Denaturation 94-96 °C 0.5-1 minutes 30-40
Annealing 45-68 °C 0.5-1 minutes
Extension 72 °C 1-2 minutes
(~1 kb/min)
Final
Extension
72 °C 10 minutes 1
Hold 4 °C Indefinitely  

 

11.    The amplified DNA can be loaded directly onto an agarose gel after the PCR is completed. It is not necessary
        to add a separate loading buffer/tracking dye.
        Note: PCR products can be purified, if desired, for applications such as sequencing with the GenElute™ PCR
        Clean-Up Kit, Catalog Number NA1020.

 

Related Products Catalog Number
PCR 96 well plates Z374903
PCR 384 well plates Z374911
Sealing mats and tape P4481; Z374938
AlumaSeal II A2350
EDTA, tripotassium salt
dihydrate
E0270
Collection Card Z719838
PCR microtubes Z374873;
Z374962; Z374881
Neutralization Solution B N3910
Mineral Oil M8662
PCR Marker P9577
Precast Agarose Gels P6097
TBE Buffer T4415;
T6400; T9525
GenElute™ PCR
Clean-Up Kit
NA1020

Troubleshooting Guide

Problem Cause Solution
Little or no PCR
product is
detected.







PCR reaction is
inhibited due to
contaminants in the
blood extract.
Use less extract or dilute the extract with water and repeat PCR. To test for inhibition, include a DNA control and/or add a known amount of template (100-500 copies) into the PCR mixture along with the blood extract.
A PCR component is
missing or degraded.
Run a positive control to insure components are functioning. A checklist is also recommended when assembling reactions.
Too few cycles are
performed.
Increase the number of cycles (5-10 additional cycles at a time).
The annealing
temperature is too high.
Decrease the annealing temperature in 2-4 °C increments.
The primers are not
designed optimally.
Confirm the accuracy of the sequence information. If the primers are less than 22 nucleotides long, try to lengthen the primer to 25-30 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%.
The denaturation
temperature is too high or too low.
Optimize the denaturation temperature by increasing or decreasing the temperature in 1 °C increments.
The denaturation time is too long or too short. Optimize the denaturation time by increasing or decreasing the time in 10 second increments.
The extension time is
too short.
Increase the extension time in 1 minute increments, especially for long templates.
The target template is complex. In most cases, inherently complex targets are due to unusually high GC content and/or secondary structure. Betaine has been reported to help amplification of high GC content templates at a concentration of 1.0-1.7 M.
Multiple products
are seen.
JumpStart Taq antibody is not working correctly. Do not use DMSO or formamide with REDExtract-N-Amp PCR ReadyMix. It can interfere with the enzyme-antibody complex. Other solvents, salts, and extremes in pH or other reaction conditions may reduce the affinity of the JumpStart Taq antibody for the Taq polymerase and thereby compromise its effectiveness.
Touchdown PCR may
be needed.
“Touchdown” PCR significantly improves the specificity of many PCR reactions in various applications. Touchdown PCR uses an annealing/extension temperature that is higher than the TM of the primers during the initial PCR cycles. The annealing/extension temperature is then reduced to the primer TM for the remaining PCR cycles. The change can be performed in a single step or in increments over several cycles.
Negative control shows a PCR
product or “false positive” results
are obtained.
Reagents are
contaminated.
Sigma recommends that a reagent blank without DNA template be included as a control in every PCR run to determine if the reagents used in extraction or PCR are contaminated with a template from a previous reaction.

AlumaSeal is a trademark of Excel Scientific, Inc.
GenElute, JumpStart, ReadyMix, and RedExtract-N-Amp are trademarks, and REDTaq is a registered trademark, of Sigma-Aldrich Co. LLC

Materials


     

References

  1. Dieffenbach, C.W., and Dveksler, G.S. (Eds.), PCR Primer: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (2003). Catalog Number Z701270
  2. Don, R.H. et al., ‘Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res., 19, 4008 (1991).
  3. Erlich, H.A. (Ed.), PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, New York (1989).
  4. Griffin, H.G., and Griffin, A.M. (Eds.), PCR Technology: Current Innovations, CRC  Press, Boca Raton, FL (1994).
  5. Innis, M.A., et al., (Eds.), PCR Strategies, Academic Press, New York (1995).
  6. Innis, M., et al., (Eds.), PCR Protocols: A Guide to Methods and Applications,  Academic Press, San Diego, California (1990).
  7. McPherson, M.J. et al., (Eds.), PCR 2: A Practical Approach, IRL Press, New York (1995).
  8. Newton, C.R. (Ed.), PCR: Essential Data, John Wiley & Sons, New York (1995).
  9. Roux, K.H. Optimization and troubleshooting in PCR. PCR Methods Appl., 4, 5185-5194 (1995).
  10. Saiki, R., PCR Technology: Principles and Applications for DNA Amplification, Stockton, New York (1989).

 

NOTICE TO PURCHASER: LIMITED LICENSE

Label License Statement

NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224 and 5,618,711. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

JumpStart and JumpStart Antibody are licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries.

RC,JC,PHC 01/13-1

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