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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
AppliChem © 2010 10/42
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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
AppliChem © 2010 11/42
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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
AppliChem © 2010 12/42
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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
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Conta-Conta-minationmination
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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)
AppliChem © 2010 22/42
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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
AppliChem © 2010 24/42
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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
AppliChem © 2010 28/42
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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:
//p l
ato.
wilm
i ngt
on.e
du/f
acu l
ty/d
troi
ke/M
o use
_ ana
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2.J P
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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
AppliChem © 2010 43/42
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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