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

2. AppliChem © 2010 2/42 There is another top address in Darmstadt!  Regeneration of Nucleic Acid Purification Matrices

3. AppliChem © 2010 3/42 There is another top address in Darmstadt! based on ExitusPlus technology for the multiple use of DNA-binding columns  maxXbond

4. AppliChem © 2010 4/42 There is another top address in Darmstadt! RG1 RG2 centrifugation degrades nucleic acids incubation (30 minutes) removes residues  maxXbond

5. AppliChem © 2010 5/42 There is another top address in Darmstadt! Quality testing I: DNA purity after regeneration  maxXbond

6. AppliChem © 2010 6/42 There is another top address in Darmstadt! Quality testing II: Binding capacity after regeneration  maxXbond

7. AppliChem © 2010 7/42 There is another top address in Darmstadt! Quality testing III: Sequencing after regenereation  maxXbond

8. AppliChem © 2010 8/42 There is another top address in Darmstadt! Quality testing IV: Stability testing after regenereation no residual RG1 on the columns linearized plasmid DNA  maxXbond

9. AppliChem © 2010 9/42 There is another top address in Darmstadt! 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

10. AppliChem © 2010 10/42 There is another top address in Darmstadt! 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

11. AppliChem © 2010 11/42 There is another top address in Darmstadt! 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

12. AppliChem © 2010 12/42 There is another top address in Darmstadt! 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

13. AppliChem © 2010 13/42 There is another top address in Darmstadt! Conta- Conta- mination mination

14. AppliChem © 2010 14/42 There is another top address in Darmstadt! o In all biological laboratories Nucleic acids are everywhere  Contamination by Nucleic Acids o In the environment

15. AppliChem © 2010 15/42 There is another top address in Darmstadt! o Cloning o PCR o Transformation o Genomic DNA Isolation o Plasmid DNA Isolation  Contamination by Nucleic Acids

16. AppliChem © 2010 16/42 There is another top address in Darmstadt! Aerosols after autoclaving  Contamination by Nucleic Acids Aerosols during pipetting Aerosols during centrifugation Personell, environment, ventilation Reagents, kits, water, plastic ware Spills during handling

17. AppliChem © 2010 17/42 There is another top address in Darmstadt! are detectable  Contamination by Nucleic Acids lead to false positive results may be harmful

18. AppliChem © 2010 18/42 There is another top address in Darmstadt! Deconta Deconta mination mination

19. AppliChem © 2010 19/42 There is another top address in Darmstadt! UV light  Decontamination of Nucleic Acids Chemical reagents Thermal destruction

20. AppliChem © 2010 20/42 There is another top address in Darmstadt!  Decontamination of Nucleic Acids no method is 100 % safe all methods destroy equipment some simply doesn‘t work

21. AppliChem © 2010 21/42 There is another top address in Darmstadt! 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)

22. AppliChem © 2010 22/42 There is another top address in Darmstadt! DescriptionHazard Symbol UN Class/PG DNA-ExitusPlus / IF-- DNA ZAPC RNase ZAPXiUN3267 8/III DNA awayXi RNase awayXiUN1824 8/II DNA-eraseXi RNase-eraseXi DNA-OFFXn RNase-OFFXn DNA-RemoverC, N LTK007(Xn) DNA-Decontamination ReagentXi Roti-NukleinsäurefreiXi bleachCUN1791 8/II  Decontamination of Nucleic Acids chemical reagents

23. AppliChem © 2010 23/42 There is another top address in Darmstadt! 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

24. AppliChem © 2010 24/42 There is another top address in Darmstadt! with / without color indicator DNA-ExitusPlus™ only active as long as not dried up simply remove by wiping off  Decontamination of Nucleic Acids

25. AppliChem © 2010 25/42 There is another top address in Darmstadt! M1 kb DNA ladder Kcontrol (sterile H 2 O) X1DNA-OFF ™ X2DNA-ZAP ™ X30.25 M NaOH, 0.5 % SDS X4DNA-Away ™ DDNA-Exitus ™ (discont.) D+DNA-ExitusPlus ™ (10 min incubation) M1 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

26. AppliChem © 2010 26/42 There is another top address in Darmstadt! X2 H 2 O 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

27. AppliChem © 2010 27/42 There is another top address in Darmstadt! Aerosols during pipetting Aerosols during centrifugation Aerosols after autoclaving Spills during handling Reagents, kits, water, plastic ware Personell, environment, ventilation  Contamination by Nucleic Acids

28. AppliChem © 2010 28/42 There is another top address in Darmstadt! Case Study I Source: http://www.uni-mainz.de/FB/Biologie/Anthropologie/MolA/English/Laboratory/Laboratory.html Ancient DNA Lab  Contamination by Nucleic Acids

29. AppliChem © 2010 29/42 There is another top address in Darmstadt! Sawing Room DNA ExtractionUV Room Sample Preparation PCR Room Lock II Lock I Ancient DNA Lab Case Study I  Contamination by Nucleic Acids

30. AppliChem © 2010 30/42 There is another top address in Darmstadt! 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]

31. AppliChem © 2010 31/42 There is another top address in Darmstadt! 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

32. AppliChem © 2010 32/42 There is another top address in Darmstadt! Tissue Sectioning http://plato.wilmington.edu/faculty/dtroike/Mouse_anatomy_012.JPG Case Study III  Contamination by Nucleic Acids

33. AppliChem © 2010 33/42 There is another top address in Darmstadt! Seed control Case 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.

34. AppliChem © 2010 34/42 There is another top address in Darmstadt! Seed control Case 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).

35. AppliChem © 2010 35/42 There is another top address in Darmstadt! Seed control Case Study IV  Contamination by Nucleic Acids Problem of unspecific PCR products caused false-negative (red arrows) or false- positive results and background (brackets).

36. AppliChem © 2010 36/42 There is another top address in Darmstadt! Seed control Case 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 of pre-PCR area, PCR workstation, as well as pipetts

37. AppliChem © 2010 37/42 There is another top address in Darmstadt!  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 Amp R gene detectable after autoclaving

38. AppliChem © 2010 38/42 There is another top address in Darmstadt!  Contamination by Nucleic Acids Autoclave-ExitusPlus™ Fig. 1: Equal volumes of either water (H 2 O) 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 H 2 O D+ M 930 bp, Amp R -Gen - M pos. M contr. H 2 O D+ Fig. 2: PCR analysis of autoclaved E. coli cultures from fig 1.

39. AppliChem © 2010 39/42 There is another top address in Darmstadt! typical contamination with RNase  Contamination by Ribonuclease (RNase)

40. AppliChem © 2010 40/42 There is another top address in Darmstadt! Lane: 1: RNase-ExitusPlus  2: empty slot 3: H 2 O 4: 10  g RNase A 5: 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)

41. AppliChem © 2010 41/42 There is another top address in Darmstadt!  Decontamination of Ribonuclease (RNase)

42. AppliChem © 2010 42/42 There is another top address in Darmstadt! DNA-ExitusPlus™ DNA-ExitusPlus™ IF surface decontamination Autoclave-ExitusPlus™ additive during autoclaving maxXbond™ maxXbond™ AX column regeneration Exi † usPlus ™ technology  Decontamination of Nucleic Acids

43. AppliChem © 2010 43/42 There is another top address in Darmstadt! 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