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Studies into the use of standard polypropylene products in life science laboratories indicate that significant amounts of DNA are lost by adherence to the walls of pipette tips, microcentrifuge tubes and microplates. Additionally, DNA fragments tend to denature when binding to the polypropylene surfaces. The wall surfaces of Axygen MAXYMUM RECOVERY products are completely free of

occlusions and cavities. This is achieved through the combination of a modification to the original polypropylene resin used in the manufacturing process, and the use of Axygen's ultra-smooth, diamond polished molds. No chemical additives are used in the manufacturing process, so no sample leaching is possible. The resulting MAXYMUM RECOVERY range of products virtually eliminates fluid retention during sample aspiration and dispensing.

Images of the internal surface of a MAXYMUM RECOVERY pipette tip. Even at the highest magnification level, the ultra-smooth surface is visibly free of occlusions and cavities which can cause sample retention and sample denaturation. Images of the internal surface of a standard polypropylene pipette tip. Occlusions and cavities can be seen on the wall surface. At the highest magnification, surface starands which cause samples to "stick", are clearly visible. Images of the internal surface of a siliconized pipette tip. Again, notice the lack of smoothness of the walls created by an inconsistent and uneven flow of silicone upon the surface. Sample retention occurs even with siliconized pipette tips.

MAXYMUM RECOVERY pipette tip after dispensing 100 l sample

Below is just a sample of Axygens MAXYMUM RECOVERY product line. Visit www.axygen.com to view the full product line, order samples or request a catalogue.
Description 10 l 10 l 10 l 20 l 20 l 50 l 100 l 150 l 200 l 300 l 1000 l 1100 l Filter Tips for P2/P10, Racked, Pre-Sterilized Filter Tips Extra Long, Racked, for P2/P10 & Eppendorf Research/Reference, Pre-Sterilized Filter Tips for Eppendorf, Racked, Pre-Sterilized Filter Tips for Eppendorf, Racked, Pre-Sterilized Filter Tips for P-20, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Filter Tips, Racked, Pre-Sterilized Cat. No. 732-0649 732-0650 732-0651 732-0936 732-0652 732-0653 732-0862 732-0654 732-0655 732-0937 732-0863 732-0935

Conventional pipette tip after dispensing 100 l sample

All the above contain 96 tips per rack (8 x 12), 960 tips per pack except 732-0863 which contains 100 tips per rack (5 x 20), 1000 tips per pack.
VWR I n t e r n a t i o n a l Issue 15 Fall 2006

Avoiding DNA loss and denaturation upon storage in plastic microtubes

Claire Gaillard and Francois Strauss
Institut Jacques Monod 2 Place Jussieu 75251 Paris 05 France

Address correspondence to: Dr. Francois Strauss, Institut Jacques Monod, 2 Place Jussieu, 75251 Paris 05, France. Internet: strauss@ijm.jussieu.fr

Key words : polypropylene tubes / DNA denaturation / DNA conformation / plastic microtubes

Summary DNA storage in polypropylene microtubes often induces loss of material and denaturation, due to DNA adsorption to tube walls. We show that, while a general problem with ordinary polypropylene tubes, even when siliconized, this does not occur with some special polypropylene tubes.

reparation of DNA fragments often requires days of exacting experiments, and it is disappointing to loose such material through faulty microfuge tubes. Tubes with obvious defects such as tight lids which refuse to open or weak walls which collapse on centrifugation do not remain on the market for long. Other defects are more difficult to detect, however, and include such subtile problems as contamination of the sample by chemicals used in the manufacturing process (1) and adsorption of DNA to the tube walls. DNA is a highly charged, hydrophilic molecule, whereas polypropylene, which is used in most microtubes, is very hydrophobic. In theory, these characteristics should reduce the interaction of DNA with tube walls and minimize loss of DNA on the tube surface. It has been observed, however, that DNA can bind to polypropylene (2) and that such interaction can induce DNA conformation changes which may extend as far as complete strand separation (3-6). This phenomenon is a particular problem with short DNA fragments and at high ionic strength. Although such interactions are known to lead to formation of interesting alternative DNA structures (ref. 7; C.G. and F.S. submitted), for most applications it is important to avoid DNA loss and denaturation. We have investigated a number of different commercially available microtubes in order to choose those which reliably neither adsorb nor denature DNA. Although we have previously observed that polyallomer tubes do not show the adsorption and denaturation observed with polyproplyene (2), the cost of polyallomer tubes is more than five-fold higher and can be prohibitive. Here we have compared polyallomer, polyethylene, and polypropylene tubes. Of the nine kinds of tubes tested, one was declared by the manufacturer to have undergone a surface treatment (siliconization) and one, which we note

here as special, was advertised to be made of pure polypropylene and to retain less DNA. Since interaction of DNA with polypropylene is particularly strong at high ionic strength, the first tests were carried out in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 containing 2.5 M NaCl. A given amount of a 32P-end labelled DNA fragment was deposited at the bottom of each tube and incubated for 16 hr at 37 using an oven to prevent condensation in the tube lid. The liquid was then removed, radioactivity remaining in the tube counted, and the percentage of adsorbed DNA calculated. Since the results often showed variability, a large number of tubes of each type were tested, and results are presented as histograms showing number of tubes as a function of the percentage of DNA adsorbed, by intervals of 5%. Results shown in Figure 1 indicate that definite trends can be discerned. All ordinary polypropylene tubes tested (a-c, e, h) showed a high percentage of DNA adsorption under these conditions. Polypropylene advertised to be silicone treated (g) showed adsorption which was not as consistent as with the untreated tubes but still reached high values. Since we observed similar results when testing polypropylene tubes which we had siliconized, we conclude that the role of silicone treatment in preventing DNA loss remains to be proven. The polyethylene tubes (i) tested bind DNA even more strongly, while, as previously observed, polyallomer tubes (d) show no adsorption. Interestingly, the special polypropylene tube (f) also showed no adsorption. Since DNA is more often used in solutions of low or moderate ionic strength, we performed similar tests in 10 mM Tris, 1 mM EDTA, pH 7.5 containing 0.1 M NaCl with incubation for 16 hr at 37. For most polypropylene tubes adsorption was relatively low, less than 5%, but some of

these tubes (a and b) continued to show binding which reached as high as 25% (data not shown). We subsequently studied the conformation of the DNA which had been subjected to these conditions. An aliquot of the solution was withdrawn from the tube after incubation, and the DNA was subjected to electrophoresis on a polyacrylamide gel to distinguish double-stranded and single-standed forms. Each sample was analyzed in triplicate, and the results are shown in Figure 2. A significant percentage of denaturation is observed with ordinary polypropylene tubes (b and c) and with siliconized polypropylene tube (g). The percentage of denaturation was lower but still detectable with polyprolylene tube (e). On the other hand, no denaturation can be seen with the polyallomer tube (d) or with the special polypropylene tube (f). In conclusion, when it is important to store a small quantity of DNA in its native double-stranded state without loss by adsorption to tube walls, the choice of tubes can be very important. Among the tubes which we have tested, ordinary polypropylene tubes cannot be recommended. In contrast, polyallomer tubes and some specially-designed polypropylene tubes show none of the problems mentioned. Other factors can be important in the choice, one being the relatively high cost of polyallomer tubes. The purity of the polymer used and the absence of surface treatment with chemicals which might remain in the tubes are two other important parameters. Unfortunately, many manufacturers are unwilling to divulge their secrets, and it is sometimes very difficult to know how the tubes are made, what additives exist in the polymer, and what surface treatment might be involved in the manufacturing process.
REFERENCES

2. Gaillard, C. and F. Strauss. 1998. Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments. Technical Tips Online T01392. 3. Belotserkovskii, B.P. and B.H. Johnston. 1996. Polypropylene tube surfaces may induce d e n a t u r a t i o n and multimerization of DNA. Science 271:222-223. 4. Gaillard, C. and F. Strauss. 1996. Polypropylene tube surfaces may induce denaturation and m u l t i m e rization of DNA - response. Science 271:223. 5. Belotserkovskii, B.P. and B.H. Johnston.1997. Denaturation and association of DNA sequences by certain polypropylene surfaces. Anal. Biochem. 251:251- 262. 6. Gaillard, C., M. Flavin, A. Woisard and F. Strauss. 1999. Association of double-stranded DNA fragments into multistranded DNA structures. Biopolymers 50:679689. 7. Gaillard, C. and F. Strauss. 1994. Association of poly(CA).poly(TG) DNA fragments into four-s t r a n d e d complexes bound by HMG1 and 2. Science 264:433-436.

1. Glossmann, H., S. Hering, A. Savchenko, W. Berger, K. Friedrich, M.L. Garcia, M.A. Goetz, J.M. Liesch, D.L. Zink and G.J. Kaczorowski. 1993. A light stabilizer (Tinuvin 770) that elutes from polypropylene plastic tubes is a potent L-type Ca(2+)-channel blocker. Proc. Natl. Acad. Sci. U. S. A. 90:9523-9527.

Figure 1. a:polypropylene 74%

DNAadsorption to tube walls in different kinds of microtubes. Nine different kinds of commercially available plastic 1.5 mL microtubes, arbitrarily labelled a-i, were purchased and tested for their capacity to adsorb DNA to their surface. A constant amount (~1 ng) of a 32 P end-labelled DNA fragment was incubated for 16 hr at 37 in 6L of 2.5 M NaCI,10 mM Tris-HCI,1 mM EDTA, pH 7.5.The percentage of DNA bound to tube walls was then determined by comparing the amount of radioactivity adsorbed and the radioactivity remaining in solution.15 to 35 tests were done with each kind of tube, and histograms were drawn with the number of tubes represented as a function of the percentage of DNA bound by intervals of 5%. For each kind of tube, the average percentage of adsorption is also indicated.

b:polypropylene 75%

c:polypropylene 77%

d:polyallomer 3%

e:polypropylene 18%

f:polypropylene 2%

g:siliconized polypropylene 52%

h:polypropylene 69% Figure 2.

Denaturation of DNAfragments as a function of the microtubes used. A given amount of labelled DNA was stored in 0.1 M NaCI, 10 mM Tris-HCI,1 mM EDTA, pH 7.5 in different kinds of 1.5 mL microtubes. After a 16 hr incubation at 37, Triton X-100 was added to 0.1 % to release any DNA bound to tube walls. Glycerol was then added to 2 % and samples were loaded on a 4% polyacrylamide gel in 6.7 mM Tris-acetate, 3.3 mM Na acetate,1 mM EDTA, pH 7.8 at 4 with buffer recirculation.On this kind of gel the single strands of the fragment can be separated from the double-stranded native form of DNA.The three DNA species are indicated on the Figure. A significant extent of denaturation and of formation of low-mobility structures is observed with some tubes, whereas in other tubes the DNA fragment remained perfectly intact in its native double-stranded form.

i:polyethylene 92%

% % DNA adsorbed

100%

Tubes tested a: Company '1' polypropylene tube,cap style x b: Company '1' polypropylene tube,cap style y c: Company '2' polypropylene tube d: Company '3' polyallomer tube e: Company '4' Axygen Standard polypropylene tube f: Company '4' Axygen MAXYMum Recovery tube g: Company '5' siliconized polypropylene tube h: Company '6' polypropylene tube i: Company '3' polyethylene tube

This article was performed by a group of scientists at the Institut Jacques Monod in Paris.The article addresses the issue of DNA loss and denaturation when using plastic tubes. Several commercially available tubes were utilized in the study. Most were manufactured from standard polypropylene. However, there were also comparisons to expensive polyallomer tubes, siliconized polypropylene tubes, polyethylene tubes, and Maxymum Recovery tubes, manufactured by Axygen Scientific.As illustrated in the study, Maxymum Recovery tubes from Axygen (f) and the polyallomer tubes (d) were far superior to the other brands compared. Maxymum Recovery tubes adsorbed less than 2% of the DNA samples, compared to a range of 69% to 92% with the competitive tubes. The polypropylene tubes (e) which adsorbed only 18% were standard, non- Maxymum Recovery tubes manufactured by Axygen Scientific. Maxymum Recovery tubes from Axygen Scientific, incorporate no additives such as silicone. Instead, the original polypropylene resin is altered, with no additives, by a proprietary formulation which is used in the manufacturing process. This process is used not only to manufacture micro centrifuge tubes, but also pipette tips, filter tips, PCR tubes, 8 strips and plates (384, 96 full skirt, 96 half skirt and 96 elevated) from Axygen Scientific. All products are available through Axygen distributors. For ordering information, please contact Axygen Scientific by telephone (510) 494-8900,by fax (510) 494-0700, by e-mail at info@axygen.com or by the internet at www.axygen.com.

Reprinted by kind permission of Claire Gaillard and Francois Strauss, Institut Jacques Monod,2 Place Jussieu, 75251 Paris 05, France

AxyGen, Inc
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