EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Reference Materials and Measurements (Geel) CERTIFIED REFERENCE MATERIAL IRMM 313 CERTIFICATE OF ANALYSIS PFGE AGAROSE PLUGS Certified value 2) SmaI digested DNA 2 fragments 1) 3 4 5 6 7 8 9 10 11 12 13 458.8 351.7 303.0 263.2 188.2 173.2 131.1 114.4 95.5 81.2 54.9 40.7 size markers, the assigned fragment sizes themselves are therefore traceable to the lambda DNA (ci857 ind 1 Sam7). 2.0 2.4 2.3 1.9 1.2 1.3 1.5 1.2 1.4 1.7 2.2 1.6 This certificate is valid for one year after purchase. Sales date: The minimum amount of sample to be used is ½ plug. Geel, January 2014 Signed: Prof. Dr. Hendrik Emons European Commission Joint Research Centre Institute for Reference Materials and Measurements Retieseweg 111 B-2440 Geel, Belgium All following pages are an integral part of the certificate. Page 1 of 5
SmaI digested DNA fragments 1) 14 15 16 Indicative Values Indicative value 2) 25.4 17.6 10.9 size markers, the assigned fragment sizes themselves are therefore traceable to the lamda DNA (ci857 ind 1 Sam7). 1.3 0.3 0.4 Page 2 of 5
Additional Material Information Indicative value 2) SmaI digested DNA 1) 1 571.7 25.4 fragments size markers, the assigned fragment sizes themselves are therefore traceable to the lamda DNA (ci857 ind 1 Sam7). PFGE pattern 1) Size marker SmaI digested IRMM-313 plug 1) Obtained with the PFGE procedure described in the instructions for use of this certificate. Page 3 of 5
DESCRIPTION OF THE MATERIAL Each unit consists of one vial containing one agarose plug for PFGE with undigested genomic DNA of Campylobacter coli CNET068 and Campylobacter jejuni CNET112 embedded. ANALYTICAL METHODS USED FOR CERTIFICATION - Enzymatic digestion - Pulsed Field Gel Electrophoresis - Polymerase Chain Reaction - Dye terminator cycle sequencing PARTICIPANTS - Animal Health and Veterinary Laboratory Agency, Surry, UK - Anses Laboratoire de Ploufragan-Plauzané, Ploufragan, France - Faculty of Veterinary Medicine, Food Institute, Skopje, Macedonia - National Veterinary Institute, Uppsala, Sweden - National Veterinary Research Institute, Pulawy, Poland - Netherlands Food and Consumer Product Safety Authority, Wageningen, Netherlands - State Veterinary and Food Institute, Dolny Kubin, Slovakia - European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium SAFETY INFORMATION The material should be handled under BSL 2 conditions. The usual laboratory safety measures apply. INSTRUCTIONS FOR USE AND INTENDED USE The main purpose of this material is to assess method performance, i.e. for checking accuracy of analytical results. Restriction enzyme digestion - Cut the plug in 2 equal pieces with a scalpel. - Incubate ½ plug in 200 µl 1x RE buffer solution, as recommended by the manufacturer, at room temperature for 15 min. - Remove the buffer using a pipette and add 200 µl RE mix, as recommended by the manufacturer, containing 40 units of SmaI. Mix by tapping gently. - Incubate at 25 ºC for 4 h. PFGE - Prepare 0.5x TBE (44.5 mm Tris-Borate; 1 mm EDTA, ph 8.3) buffer from 5x TBE buffer by diluting in Type I water. - Fill the PFGE chamber with sufficient 0.5x TBE buffer, remove air bubbles trapped in the tubing, Set the cooler at 14 C and circulate the buffer for 1 h before use. - Prepare 150 ml 1 % SKG agarose in 0.5x TBE and keep at 50 ºC until use - Place the plug pieces on the comb as far to the bottom of the teeth as possible and fix them onto the comb by pipetting a few drops 1 % SKG agarose around the plugs. Place an IRMM-313 plug piece on the outside lanes of each gel and at least next to every 4 th unknown sample. - Assemble the casting stand and place it on a levelling table. It is important that the casting stand is completely horizontal to have the same gel thickness all over the gel. - put the comb, with the plugs attached in place. - Pour in the 1 % SKG agarose making sure not to move the plugs. Remove any air bubbles. Allow the gel to solidify for 30 to 45 min. - Carefully remove the comb from the gel. - Seal the resulting wells with 1 % SKG agarose. - Take the gel and the black plate out of the casting stand. Remove any agarose from the bottom of the plate. - Place the plate with the gel in the electrophoresis chamber, be sure that it fits tightly in the frame. Close the chamber, check the tubing for bubbles and check that the cooler is working. - Start the run with running conditions: Initial switch time: 0.5 s Final switch time: 40 s Gradient: 5 V/cm Angle: 120º - Let run for 22.5 h - Stain the gel 30 min in 400 ml 1 µg/ml ethidium bromide in Type I water. - De-stain for 2x 20 min in 400 ml Type I water. - Photograph the gel over UV-source using a UV orange filter. - Analyse the gel using a dedicated software. Page 4 of 5
STORAGE The materials are recommended to be stored at 4 C. The plug pieces are recommended to be used on the same gel. However, the European Commission cannot be held responsible for changes that happen during storage of the material at the customer's premises, especially of opened samples. LEGAL NOTICE Neither IRMM, its subsidiaries, its contractors nor any person acting on their behalf, (a) make any warranty or representation, express or implied that the use of any information, material, apparatus, method or process disclosed in this document does not infringe any privately owned intellectual property rights; or (b) assume any liability with respect to, or for damages resulting from, the use of any information, material, apparatus, method or process disclosed in this document save for loss or damage arising solely and directly from the negligence of IRMM or any of its subsidiaries. NOTE A technical report on the production of IRMM-313 is available on the internet (www.irmm.jrc.be). A paper copy can be obtained from IRMM on request. European Commission Joint Research Centre Institute for Reference Materials and Measurements (IRMM) Retieseweg 111, B - 2440 Geel (Belgium) Telephone: +32-(0)14-571.722 - Telefax: +32-(0)14-590.406 Page 5 of 5