AppliChem © 2010 1/42 There is another top address in Darmstadt! ExitusPlus™ Technology ...

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AppliChem © 2010 1/42 There is another top address in Darmstadt! ExitusPlus™ Technology Contamination by Nucleic Acids Decontamination of Nucleic Acids RNase-ExitusPlus maxXbond

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Page 1: AppliChem © 2010 1/42 There is another top address in Darmstadt! ExitusPlus™ Technology Contamination by Nucleic Acids Decontamination of Nucleic Acids.

AppliChem © 2010 1/42

There is another top address in Darmstadt!

ExitusPlus™ Technology

Contamination by Nucleic Acids

Decontamination of Nucleic Acids

RNase-ExitusPlus

maxXbond

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Regeneration of Nucleic Acid Purification Matrices

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• based on ExitusPlus technology

• for the multiple use of DNA-binding columns

maxXbond

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RG1

RG2

centrifugation

degrades nucleic acids

incubation(30 minutes)

removes residues

maxXbond

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• Quality testing I: DNA purity after regeneration

maxXbond

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• Quality testing II: Binding capacity after regeneration

maxXbond

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• Quality testing III: Sequencing after regenereation

maxXbond

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• Quality testing IV: Stability testing after regenereationno residual RG1 on the columns

linearized plasmid DNA

maxXbond

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For the small ones (Mini columns) pure Silica matrices

... and the larger ones (Midi, Maxi, Mega, Giga)

AX = Anion eXchanger

• The small difference with the big consequences:

maxXbond

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• Indication of DNA contaminations of new columns

van der Zee & Crielaad (2002) Journal of Clinical Microbiology 40, 1126

Evans et al. (2003) Journal of Clinical Microbiology, 41, 3452-3453

Remco et al. (2004) EMS Immunology and Medical Microbiology 42, 249-253

Detection of contamination with Legionella -DNA

DNA contamination leads to false-positive findings

maxXbond

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• Refill Buffer Set for maxXbond regenerated columns maxXmore

AppliChem's 5* Star buffer set (S1 - S5)optimized for maxXbond-regenerated columns

- all buffers ready-to-use- no edition of ethanol required- storage at ambient temperature

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• Refill Buffer Set for maxXbond AX regenerated columns

maxXmore AX

AppliChem's 6* Star buffer set (SM1 - SM6)optimized for maxXbond AX-regenerated columns

- all buffers ready-to-use- no addition of ethanol required- storage at ambient temperature

Page 13: AppliChem © 2010 1/42 There is another top address in Darmstadt! ExitusPlus™ Technology Contamination by Nucleic Acids Decontamination of Nucleic Acids.

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Conta-Conta-minationmination

Page 14: AppliChem © 2010 1/42 There is another top address in Darmstadt! ExitusPlus™ Technology Contamination by Nucleic Acids Decontamination of Nucleic Acids.

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o In all biological laboratories

• Nucleic acids are everywhere

Contamination by Nucleic Acids

o In the environment

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o Cloning

o PCR

o Transformation

o Genomic DNA Isolation

o Plasmid DNA Isolation

Contamination by Nucleic Acids

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• Aerosols after autoclaving

Contamination by Nucleic Acids

• Aerosols during pipetting

• Aerosols during centrifugation

• Personell, environment, ventilation

• Reagents, kits, water, plastic ware

• Spills during handling

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• are detectable

Contamination by Nucleic Acids

• lead to false positive results

• may be harmful

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DecontaDecontaminationmination

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• UV light

Decontamination of Nucleic Acids

• Chemical reagents

• Thermal destruction

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Decontamination of Nucleic Acids

• no method is 100 % safe

• all methods destroy equipment

• some simply doesn‘t work

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o strong mineralic acids and bases

except for bleach not very effective

Decontamination of Nucleic Acids

• chemical reagents

o peroxides

o azides

o hypochlorite ("bleach")

o organic solvents (toxic fumes)

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Description Hazard Symbol UN Class/PG

DNA-ExitusPlus / IF - -

DNA ZAP C

RNase ZAP Xi UN3267 8/III

DNA away Xi

RNase away Xi UN1824 8/II

DNA-erase Xi

RNase-erase Xi

DNA-OFF Xn

RNase-OFF Xn

DNA-Remover C, N

LTK007 (Xn)

DNA-Decontamination Reagent Xi

Roti-Nukleinsäurefrei Xi

bleach C UN1791 8/II

Decontamination of Nucleic Acids

• chemical reagents

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• aqueous solution* or powder

• all components biodegradable

• synergistic action of biomolecules and a mild acid

• low concentration of all ingredients

• noncorrosive, nontoxic

• very effective, especially at elevated temperatures

Decontamination of Nucleic Acids

* contains < 10 % of alcohol

Exi†usPlus™ technology

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• with / without color indicatorDNA-ExitusPlus™ only

• active as long as not dried up

• simply remove by wiping off

Decontamination of Nucleic Acids

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M 1 kb DNA ladder

K control (sterile H2O)

X1 DNA-OFF™

X2 DNA-ZAP™

X3 0.25 M NaOH, 0.5 % SDS

X4 DNA-Away™

D DNA-Exitus™ (discont.)

D+ DNA-ExitusPlus™

D+ DNA-ExitusPlus™

(10 min incubation)

M 1 kb DNA ladder

M K X1 X2 X3 X4 D D+ D+ M

- 500 bp

- 1000 bp

- 50 bp

Incubation 3 min 10 min

Decontamination of Nucleic Acids• Quality control

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X2 H2O X3 D+ X4

silver with gold cover

silver

stainless steel

aluminium

copper

zinc

X2, X3, X4: competitors, D+: DNA-ExitusPlus

Decontamination of Nucleic Acids• Quality control

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• Aerosols during pipetting

• Aerosols during centrifugation

• Aerosols after autoclaving

• Spills during handling

• Reagents, kits, water, plastic ware

• Personell, environment, ventilation

Contamination by Nucleic Acids

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Case Study I

Source: http://www.uni-mainz.de/FB/Biologie/Anthropologie/MolA/English/Laboratory/Laboratory.html

Ancient DNA Lab

Contamination by Nucleic Acids

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Sawing Room

DNA ExtractionUV Room

Sample Preparation

PCR Room

Lock II

Lock I

Ancient DNA Lab

Case Study I Contamination by Nucleic Acids

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• Measures to prevent DNA contamination:

o Locks (sealed rooms)

Case Study I Contamination by Nucleic Acids

Ancient DNA Lab

o Separation of pre-PCR and PCR areas: separate rooms

o regular decontamination with DNA-ExitusPlus IF of surfaces and equipment.decontamination of rooms with sodium hypochlorite[e.g. Chlorox]

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State Police Departments

customer statement published as interview (LKA State of Thuringia) in labor&more 0107

• DNA decontamination of 1. surgical instruments & 2. PCR workstations

Case Study II Contamination by Nucleic Acids

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Tissue Sectioning

http:

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wilm

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on.e

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Case Study III Contamination by Nucleic Acids

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Seed controlCase Study IV

Contamination by Nucleic Acids

DSV laboratories analyze rape DNA from seeds in high-throughput screening, up to 36 x 96 well plates per day.

Requirements: DNA isolation from single rape seeds (1 - 3 mm diameter); therefore very small amounts of starting material of PCR templates for subsequent PCR. Highest priority: prevention of cross-contamination by DNA.

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Seed controlCase Study IV

Contamination by Nucleic Acids

Multiplex-PCR for identification of species with 3 specific PCR products (arrows).

Appearance of increasing numbers of unspecific PCR products (brackets).

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Seed controlCase Study IV

Contamination by Nucleic Acids

Problem of unspecific PCR products caused false-negative (red arrows) or false-positive results and background (brackets).

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Seed controlCase Study IV

Contamination by Nucleic Acids

Measures to prevent contamination:

1. Removal of the autoclave from the neighbouring laboratory

2. Washing out the autoclave, refrigerator and table-top centrifuges with DNA-ExitusPlus IF

3. Regular decontamination with DNA-ExitusPlus IF ofpre-PCR area, PCR workstation, as well as pipetts

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Contamination by Nucleic Acids

• Autoclave-ExitusPlus™

o autoclaves are a main source of contamination

o vapours contain nucleic acids

o incomplete degradation of nucleic acids during autoclaving

o the complete AmpR gene detectable after autoclaving

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Contamination by Nucleic Acids

• Autoclave-ExitusPlus™

Fig. 1: Equal volumes of either water (H2O) or Autoclave-ExitusPlus (D+), respectively, were added to 50 ml cultures of recombinant E. coli and autoclaved at 120°C and 1.2 bar for 20 min.

- 500 bp

- 1000 bp

- ~20 bp

H2O D+ M

930 bp, AmpR-Gen -

M pos. M contr. H2O D+

Fig. 2: PCR analysis of autoclaved E. coli cultures from fig 1.

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typical contamination with RNase

Contamination by Ribonuclease (RNase)

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Lane:1: RNase-ExitusPlus

2: empty slot3: H2O

4: 10 g RNase A5: empty slot

C: untreated total RNA

1, 3, 4 = plus 10 µg RNase A

1, 3, 4 = treatment as indicated

1, 3, 4 = plus 5 µg RNA

4 = plus 10 µg RNase A

Decontamination of Ribonuclease (RNase)

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Decontamination of Ribonuclease (RNase)

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DNA-ExitusPlus™

DNA-ExitusPlus™ IF

surfacedecontamination

Autoclave-ExitusPlus™ additive duringautoclaving

maxXbond™

maxXbond™ AX

columnregeneration

Exi†usPlus™ technology

Decontamination of Nucleic Acids

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• Best price!

• Best activity!

• Most pack sizes (100 ml - 1 L spray; refills)

• Best compatibility! Not harmful!

• Many potential customers

• Consumption 1 L / 10 m²

Decontamination: more sales arguments


Nucleic Acids and Protein Synthesis. What are nucleic acids?

Nucleic Acids and Protein Synthesis. What are nucleic acids?

Biochemistry 3070 – Nucleic Acids 1 Nucleic Acids Biochemistry 3070.

Biochemistry 3070 – Nucleic Acids 1 Nucleic Acids Biochemistry 3070.

Nucleotides and Nucleic Acids. Definitions Nucleic acids are polymers of nucleotides In eukaryotic cells nucleic acids are either: Deoxyribose nucleic.

Nucleotides and Nucleic Acids. Definitions Nucleic acids are polymers of nucleotides In eukaryotic cells nucleic acids are either: Deoxyribose nucleic.

NUCLEIC ACIDS INTRODUCTION - oswego.edukadima/CHE525/NUCLEIC ACIDS_INTRODUCTI… · NUCLEIC ACIDS An INTRODUCTION Two classes of Nucleic Acids • Deoxynucleic Acids (DNA) – Hereditary

NUCLEIC ACIDS INTRODUCTION - oswego.edukadima/CHE525/NUCLEIC ACIDS_INTRODUCTI… · NUCLEIC ACIDS An INTRODUCTION Two classes of Nucleic Acids • Deoxynucleic Acids (DNA) – Hereditary

Nucleic Acids Nucleic acids - Weeblymarshallwong.weebly.com/uploads/7/.../nucleic_acids... · Nucleic acids AP Biology 2006-2007 Nucleic Acids Information storage AP Biologyproteins

Nucleic Acids Nucleic acids - Weeblymarshallwong.weebly.com/uploads/7/.../nucleic_acids... · Nucleic acids AP Biology 2006-2007 Nucleic Acids Information storage AP Biologyproteins

Nucleic Acids:

Nucleic Acids:

Nucleic Acids

Nucleic Acids

Ch 5 Nucleic Acids excerpt - philipdarrenjones.comphilipdarrenjones.com/web_documents/ch_5_nucleic_acids_excerpt.pdf · Ch 5 (excerpt) Nucleic Acids Nucleic Acids •Store ... proteins

Ch 5 Nucleic Acids excerpt - philipdarrenjones.comphilipdarrenjones.com/web_documents/ch_5_nucleic_acids_excerpt.pdf · Ch 5 (excerpt) Nucleic Acids Nucleic Acids •Store ... proteins

CHAPTER 2 STRUCTURES OF NUCLEIC ACIDS nucleic acids

CHAPTER 2 STRUCTURES OF NUCLEIC ACIDS nucleic acids

Nucleic acids 2006-2007 Nucleic Acids Information storage.

Nucleic acids 2006-2007 Nucleic Acids Information storage.

nucleic acids

nucleic acids

A72 Contaminations by Nucleic Acids - dnagdansk.com · Contaminations by Nucleic Acids ... AppliChem stands for a continuous control of the product quality during ... complementary

A72 Contaminations by Nucleic Acids - dnagdansk.com · Contaminations by Nucleic Acids ... AppliChem stands for a continuous control of the product quality during ... complementary

Nucleic acids Carbohydrates Lipids Proteins Nucleic acids.

Nucleic acids Carbohydrates Lipids Proteins Nucleic acids.

21 - 1CH110 – Nucleic Acids Nucleic acids Nucleic acids Nucleic acids: –Maintain genetic information –Determine Protein Synthesis DNAdeoxy DNA= deoxyribonucleic.

21 - 1CH110 – Nucleic Acids Nucleic acids Nucleic acids Nucleic acids: –Maintain genetic information –Determine Protein Synthesis DNAdeoxy DNA= deoxyribonucleic.