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Life Science Quarterly Summer 2002

Extract-N-Amp Faqs

 Life Science
Quarterly
 A TECHNICAL APPLICATION NEWSLETTER VOL 3   ISSUE 2   Summer 2002

 Featured Article Frequently Asked Questions

As a major Life Science supplier, Sigma-Aldrich has spent years helping customers with numerous application and technique inquiries. By using some of the information gained from these customer contacts, we present some of our Frequently Asked Questions (FAQs). These FAQs will hopefully serve as a guide to help identify and resolve some of your concerns.

Extract-N-AmpTM Blood Kit

Can I screen for viral or bacterial targets?

Probably not. Most infectious organisms are present at fairly low levels in blood. With Extract-N-Amp Blood, only 0.1 µl of blood is analyzed by PCR. This amount of blood contains sufficient host DNA (1-3 ng) for PCR amplification. However, you would need > 10 organisms/ml of blood to detect virus or bacteria.

Can the isolated DNA be manipulated by means other than PCR – such as restriction digestion or (more importantly) PCR sequencing?

The DNA prepared with Extract-N-Amp is not adequately purified for most downstream applications – in fact, it isn’t purified at all. PCR is so sensitive that very little DNA, and hence, very little of the accompanying impurities are added to the reaction. The amount of DNA generally used for restriction digests and visualization on ethidium bromide-stained gels will likely contain enough impurities to inhibit a restriction enzyme. On the other hand, we have obtained good sequencing data from PCR products generated with the Extract-N-Amp Blood Kit. For an example, (See Figure 5)

How can samples be collected and stored prior to putting them into the procedure? Is fresh blood better than frozen or otherwise stored – or do “aged” cells lyse better?

The Extract-N-Amp Blood Kit works well with fresh, frozen, and aged blood. (See Figure 2)

Can I store blood in lysis buffer only?

Probably, yes. However, we have not tested storage in lysis solution alone.

Is it necessary to include EDTA or other anticoagulants?

Yes. If you do not collect the blood into an anticoagulant, the cells will coagulate and form a clot. It is likely the lysis solution will release DNA from a clot, but we have not tested this and would not know what volume of the reagent to recommend.

How long can the extracted/neutralized DNA be stored?

The neutralized extracts are stable for at least 6 months at 4 °C (See Figure 3) . Since extracts at 37 °C for 6 months also showed no loss of PCR-detected DNA, we estimate that the extracts will be stable for nearly 5 years at 4 °C.

What is the red dye for? Will it affect automated sequencing?

The red dye is for tracking migration during agarose gel electrophoresis. This dye exhibts negligible absorption at the excitation wavelength. Absortion is undetectable after routine post sequencing reaction clean-up.

What is the largest/smallest DNA that can be extracted and amplified using this kit?

We have amplified PCR products as small as 125 bp and as large as 5 kb.

Can TA cloning be used on the products?

Yes! (See Figure 4)

Can RNA from the samples be used in RT-PCR?

No. RNA is degraded during the cell lysis procedure.

Will this kit work with real-time quantitative PCR?

Absolutely! The data demonstrating extract stability in (Figure 3) were obtained by real-time quantitative PCR on an ABI PRISM™ 7700 (Perkin Elmer/Applied BioSystem).

For advice and information on our products, contact Sigma Technical Services at 1-800-325-5832 or by email at techserv@sial.com


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