Conventionalpipette tip after dispensing 100 µl sample

VWR I n t e r n a t i o n a l Issue 15 F a l l 2 0 0 6 3

Studies into the use of standard polypropyleneproducts in life science laboratories indicatethat significant amounts of DNA are lost byadherence to the walls of pipette tips,microcentrifuge tubes and microplates.Additionally, DNA fragments tend to denaturewhen binding to the polypropylene surfaces.The wall surfaces of Axygen MAXYMUMRECOVERY products are completely free of

occlusions and cavities. This is achievedthrough the combination of a modification tothe original polypropylene resin used in themanufacturing process, and the use ofAxygen's ultra-smooth, diamond polishedmolds. No chemical additives are used in themanufacturing process, so no sample leachingis possible.The resulting MAXYMUM RECOVERY range ofproducts virtually eliminates fluid retentionduring sample aspiration and dispensing.

MAXYMUM RECOVERY PlasticsUltra-Smooth Surfaces Optimize Sample Handling Performance

For more information on these products contact your local VWR sales office,send an e-mail to vwrbiomarke@eu.vwr.comor visit our website www.vwr.com

Images of the internal surface of a MAXYMUMRECOVERY pipette tip. Even at the highest magni-fication level, the ultra-smooth surface is visiblyfree of occlusions and cavities which can causesample retention and sample denaturation.

Images of the internal surface of a standardpolypropylene pipette tip. Occlusions and cavitiescan be seen on the wall surface. At the highestmagnification, surface starands which causesamples to "stick", are clearly visible.

Images of the internal surface of a siliconizedpipette tip. Again, notice the lack of smoothnessof the walls created by an inconsistent anduneven flow of silicone upon the surface. Sampleretention occurs even with siliconized pipette tips.

MAXYMUM RECOVERYpipette tip after dispensing 100 µl sample

Description Cat. No.

10 µl Filter Tips for P2/P10, Racked, Pre-Sterilized 732-064910 µl Filter Tips Extra Long, Racked, for P2/P10 & Eppendorf

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1000 µl Filter Tips, Racked, Pre-Sterilized 732-08631100 µl Filter Tips, Racked, Pre-Sterilized 732-0935

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Avoiding DNA loss and denaturation upon storage inplastic microtubesClaire Gaillard and Francois Strauss

Institut Jacques Monod2 Place Jussieu75251 Paris 05France

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 / plasticmicrotubes

SummaryDNA storage in polypropylene microtubes often induces loss of material and denatu-ration, due to DNA adsorption to tube walls. We show that, while a general problem withordinary polypropylene tubes, even when siliconized, this does not occur with somespecial polypropylene tubes.

P reparation of DNA fragments often requires days ofexacting experiments, and it is disappointing toloose such material through faulty microfuge

tubes. Tubes with obvious defects such as tight lids whichrefuse to open or weak walls which collapse on centrifu-gation do not remain on the market for long. Other defectsare more difficult to detect, however, and include suchsubtile problems as contamination of the sample bychemicals used in the manufacturing process (1) andadsorption of DNA to the tube walls.

DNA is a highly charged, hydrophilic molecule, whereaspolypropylene, which is used in most microtubes, is veryhydrophobic. In theory, these characteristics should reducethe interaction of DNA with tube walls and minimize lossof DNA on the tube surface. It has been observed,h o w e v e r, that DNA can bind to polypropylene (2) and thatsuch interaction can induce DNA conformation changeswhich may extend as far as complete strand separation(3-6). This phenomenon is a particular problem with shortDNA fragments and at high ionic strength. Although suchinteractions are known to lead to formation of interestingalternative DNA structures (ref. 7; C.G. and F.S. submitted),for most applications it is important to avoid DNA loss andd e n a t u r a t i o n .

We have investigated a number of different commerciallyavailable microtubes in order to choose those whichreliably neither adsorb nor denature DNA. Although wehave previously observed that polyallomer tubes do notshow the adsorption and denaturation observed withpolyproplyene (2), the cost of polyallomer tubes is morethan five-fold higher and can be prohibitive. Here we havecompared polyallomer, polyethylene, andpolypropylene tubes. Of the nine kinds of tubes tested, onewas declared by the manufacturer to have undergone asurface treatment (siliconization) and one, which we note

here as “special,” was advertised to be made of purepolypropylene and to retain less DNA.

Since interaction of DNA with polypropylene is partic-ularly strong at high ionic strength, the first tests werecarried out in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5containing 2.5 M NaCl. A given amount of a 3 2P - e n dlabelled DNA fragment was deposited at the bottom ofeach tube and incubated for 16 hr at 37° using an oven toprevent condensation in the tube lid. The liquid was thenremoved, radioactivity remaining in the tube counted, andthe percentage of adsorbed DNA calculated. Since theresults often showed variability, a large number of tubesof each type were tested, and results are presented ashistograms showing number of tubes as a function of thepercentage of DNA adsorbed, by intervals of 5%.

Results shown in Figure 1 indicate that definite trends canbe discerned. All ordinary polypropylene tubes tested (a-c,e, h) showed a high percentage of DNA adsorption underthese conditions. Polypropylene advertised to be siliconetreated (g) showed adsorption which was not asconsistent as with the untreated tubes but still reachedhigh values. Since we observed similar results whentesting polypropylene tubes which we had siliconized, weconclude that the role of silicone treatment in preventingDNA loss remains to be proven. The polyethylene tubes (i)tested bind DNA even more strongly, while, as previouslyobserved, polyallomer tubes (d) show no adsorption.I n t e r e s t i n g l y, the “special” polypropylene tube (f) alsoshowed no adsorption.Since DNA is more often used in solutions of low ormoderate ionic strength, we performed similar tests in 10mM Tris, 1 mM EDTA, pH 7.5 containing 0.1 M NaCl withincubation for 16 hr at 37°. For most polypropylene tubesadsorption was relatively low, less than 5%, but some of

these tubes (a and b) continued to show binding whichreached as high as 25% (data not shown). We subsequentlystudied the conformation of the DNA which had beensubjected to these conditions. An aliquot of the solution waswithdrawn from the tube after incubation, and the DNA wassubjected to electrophoresis on a polyacrylamide gel todistinguish double-stranded and single-standed forms. Eachsample was analyzed in triplicate, and the results are shown inFigure 2. A significant percentage of denaturation is observedwith ordinary polypropylene tubes (b and c) and with siliconizedpolypropylene tube (g). The percentage of denaturation waslower but still detectable with polyprolylene tube (e). On theother hand, no denaturation can be seen with the polyallomertube (d) or with the “special” polypropylene tube (f).

In conclusion, when it is important to store a small quantityof DNA in its native double-stranded state without loss byadsorption to tube walls, the choice of tubes can be veryimportant. Among the tubes which we have tested, ordinarypolypropylene tubes cannot be recommended. In contrast,polyallomer tubes and some specially-designed polypropylenetubes show none of the problems mentioned. Other factorscan be important in the choice, one being the relatively highcost of polyallomer tubes. The purity of the polymer used andthe absence of surface treatment with chemicals which mightremain in the tubes are two other important parameters.U n f o r t u n a t e l y, many manufacturers are unwilling to divulgetheir secrets, and it is sometimes very difficult to know howthe tubes are made, what additives exist in the polymer, andwhat surface treatment might be involved in the manufac-turing process.

R E F E R E N C E S

1 . Glossmann, H., S. Hering, A. Savchenko, W. Berger, K.Friedrich, M.L. Garcia, M.A. Goetz, J.M. Liesch, D.L. Zinkand G.J. Kaczorowski. 1993. A light stabilizer (Tinuvin7 70) that elutes from polypropylene plastic tubes is apotent L-type Ca(2+)-channel blocker.Proc. Natl. Acad. Sci. U. S. A. 9 0 : 9 5 2 3 - 9 5 2 7 .

2 . Gaillard, C. and F. Strauss. 1998. Avoiding adsorption ofDNA to polypropylene tubes and denaturation of shortDNA fragments. Technical Tips OnlineT 01 3 9 2 .

3 . Belotserkovskii, B. P. and B.H. Johnston. 1996.Polypropylene tube surfaces may induced e n a t u r a t i o n and multimer ization of DNA.Science 271 : 2 2 2 - 2 2 3 .

4 . Gaillard, C. and F. Strauss. 1996. Polypropylene tubesurfaces may induce denaturation and m u l t i m e r-ization of DNA - response. Science 271 : 2 2 3 .

5 . Belotserkovskii, B. P. and B.H. Johnston.1997.Denaturation and association of DNA sequences bycertain polypropylene surfaces. Anal. Biochem. 2 51 : 2 51- 262.

6 . Gaillard, C., M. Flavin, A. Woisard and F. Strauss. 1999.Association of double-stranded DNA fragments intomultistranded DNA structures. Biopolymers 50:679-6 8 9 .

7 . Gaillard, C. and F. Strauss. 1994. Association ofp o l y ( C A ) . p o l y ( TG) DNA fragments into four-s t r a n d e dcomplexes bound by HMG1 and 2. Science 264:433-436.

Figure 1.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 andtested for their capacity to adsorb DNA to their surface. A constant amount (~1 ng) of a 32P end-labelled DNAfragment was incubated for 16 hr at 37° in 6µL of 2.5 M NaCI,10 mM Tris-HCI,1 mM EDTA, pH 7.5.The percentageof DNA bound to tube walls was then determined by comparing the amount of radioactivity adsorbed and theradioactivity remaining in solution.15 to 35 tests were done with each kind of tube, and histograms were drawn withthe number of tubes represented as a function of the percentage of DNA bound by intervals of 5%. For each kindof tube, the average percentage of adsorption is also indicated.

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 7.5in different kinds of 1.5 mL microtubes. After a 16 hr incubation at 37°, Triton X-100 was addedto 0.1 % to release any DNA bound to tube walls. Glycerol was then added to 2 % and sampleswere loaded on a 4% polyacrylamide gel in 6.7 mM Tris-acetate, 3.3 mM Na acetate,1 mMEDTA, pH 7.8 at 4° with buffer recirculation.On this kind of gel the single strands of the fragmentcan be separated from the double-stranded native form of DNA.The three DNA species areindicated on the Figure. A significant extent of denaturation and of formation of low-mobilitystructures is observed with some tubes, whereas in other tubes the DNA fragment remainedperfectly intact in its native double-stranded form.

a:polypropylene 74%

b:polypropylene

75%

c:polypropylene

77%

d:polyallomer

3%

e:polypropylene

18%

f:polypropylene

2%

g:siliconized

polypropylene

52%

h:polypropylene

69%

i:polyethylene

92%

% 100%% DNA adsorbed

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

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

AxyGen, Inc33170 Central Avenue, Union City, California 94587, United StatesTel:(510) 494-8900 Fax:(510) 494-0700 Email :jaz@axygen.com Web :www.axygen.com

This article was performed by a group of scientists at the Institut Jacques Monod in Paris.The article addresses the issue ofDNA loss and denaturation when using plastic tubes. Several commercially available tubes were utilized in the study. Mostwere 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.Asillustrated in the study, Maxymum Recovery tubes from Axygen (f) and the polyallomer tubes (d) were far superior to the otherbrands compared. Maxymum Recovery tubes adsorbed less than 2% of the DNA samples, compared to a range of 69% to92% with the competitive tubes. The polypropylene tubes (e) which adsorbed only 18% were standard, non- MaxymumRecovery tubes manufactured by Axygen Scientific.

Maxymum Recovery tubes from Axygen Scientific, i n c o r p o r ate no additives such as silicone. I n s t e a d , the originalpolypropylene 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 stripsand 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 bytelephone (510) 494-8900,by fax (510) 494-0700, by e-mail at info@axygen.com or by the internet at www.axygen.com.