User Guide
Contents
1. 5 4 Centrifuge the sample at soeed between 6000 and 10000 x g for 10 minutes 5 5 Transfer the amount of aqueous phase indicated in Table 2 to a 2 or to a 15 ml tube on the basis of your sample Note the upper phase is aqueous in samples containing fats or oils the aqueous phase lies in the intermediate phase 5 6 Add the amount of Purification Solution indicated in Table 2 and shake vigorously 1 minute then centrifuge the sample between 10000 and 11000 x g for 5 minutes Note the supernatant must be clear If not repeat the centrifugal step Proceed to the section 6 of this protocol User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15 7 ENER N Advanced Transfer Technologies 6 Transfer into DNA binding column and elution 6 1 Transfer the amount of supernatant indicated in Table 1 or in Table 2 to a polypropylene tube containing 5 ml of Buffer T avoid to pick up the proteins located in the intermediate phase For matrices reported in RED in Table 1 gather the aliquots of supernatant previously split and transfer into a tube containing 7 5 ml of Buffer T 6 2 Mix gently by inversion to avoid DNA shearing stress Note Buffer T contains a pH indicator that turns from yellow to red when pH gt 7 5 In case the colour turns to red acidify the extract by adding glacial acetic acid CH COOH drop by drop to correct the pH value until the colour of the buffer turns to yellow 6 3 If required by your ma2. Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers Each step of sample preparation grinding transferring weighing etc must be done according to GLP so that chance of cross contamination between samples is minimized It is recommended to use disposable equipment when possible If the food samples are not in a powdered or granular form they should be processed grinded or blended before DNA extraction The majority of DNA extraction methods supports from 20 to 50 mg of starting material ION Force DNA Extractor FAST allows processing up to 20 grams of starting material in order to maximize sample s lot representation Once the sample has been pulverized homogenized it can be weighed and the appropriate amount extracted according to DNA extraction method selected Refer to manufacturer user manual for extraction procedure details User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15 4 ENER N Advanced Transfer Technologies 4 DNA Extraction from Food and Feed 1 Grind and or homogenize the sample Transfer into a 50 ml tube the requested quantity as indicated in Table 1 Matrix Weight 0 2 g Al
3. ENER N Advanced Transfer Technologies ION FORCE DNA EXTRACTOR FAST Cat N EXDOOL User Guide Rev 32 of 06 08 15 ENER N Advanced Transfer Technologies 1 Introduction ION Force FAST is a DNA extraction kit developed to fulfil the demands of the molecular biologist working in the food and feed field This kit has a very flexible protocol that allows DNA recovery from a wide range of matrices In this user manual protocols covering the majority of the applications are reported Products available E Pe lon Force DNA Extractor kit 100 rx lon Force DNA Extractor SOLUTION A 2000 ml EXD004 lon Force DNA Extractor SOLUTION D 150 ml ETS lon Force DNA Extractor BUFFER T 750 ml EXD006 lon Force DNA Extractor BUFFER P 50 ml EXD007 lon Force DNA Extractor COLUMNS gt Cy Adapters for BOX VACUUM pack 12 30100A EDNA Extraction BOX VACUUM Plastic User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15 2 C SENERON 2 lon Force DNA Extractor FAST 2 1 Kit Content The kit 100 test contains the following reagents Product Quantity Solution A 4x 500 ml Purification Solution 1x 100ml Columns 100 pcs Collection Tubes 100 pcs Buffer T 3 x 250 ml Buffer P concentrate 1x50 ml Solution D 1x20 ml Buffer P concentrate should be diluted by adding 200 ml absolute ethanol to obtain 250 ml of ready to use solution Label the bottle after adding 2 2 Storage amp Expiry information Expiry date s
4. ee date on the packaging product validity refers to the product kept intact in its original packaging Store at room temperature Do not freeze the reagents or refrigerate below 8 C 2 3 Warranty and responsibility Generon s r l guarantees the buyer exclusively concerning the quality of reagents and of the components used to produce the kit In case of defective products Generon s r l will replace it with a new one Generon s r l is not responsible and cannot anyway be considered responsible or jointly responsible for possible damages resulting from the utilization of the product by the user User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15 3 ENER N Advanced Transfer Technologies 3 Materials and equipments needed Material Equipment Chemicals n esane Tubes 50 ml and 15 ml Adapters for columns and syringes EXD010 A Plastic spoons Technical balance or similar sensitivity 0 01 g 10 ml syringes without needle and cellulose acetate filters 0 45 um pore size Graduated cilinder 50 ml Moulinex homogenizer or equivalent Centrifuge with rotors for 1 5 2 ml microtubes and 50 ml tubes range within 100 11200 xg or rcf Thermal Water Bath or Block heated up to 85 C 2 C Pipette sets Pipette tips Barrier Tube rack for 1 5 ml tubes 2 0 and 1 5 ml micro tubes DNA Extraction VACUUM BOX Vacuum pump or Venturi meter Source Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers
5. he intermediate phase 4 5 Add 0 8 ml of Purification Solution and shake vigorously 1 minute then centrifuge the Sample between 10000 and 11000 x g for 5 minutes Note the supernatant must be clear If not repeat the centrifugal step Proceed to the section 6 of this protocol User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15 6 Gpeneren 5 Specific DNA extraction from particular matrix 5 1 Transfer into a 15 or 50 ml tube the requested quantity as indicated in Table 2 Weight Incubation Aqueousphaseto Purification Aliquot of mou 0 2 go ml oton iA time be transferred Solution supernatant Animal M ilk 50 ml 5 ml 60 min 4 ml 4 ml 3 5 ml Enrichment media 1ml 9 ml 15 min 1ml 0 8 ml 0 9 ml Filters swabs 1 unit 20 ml 15 min Not necessary Not necessary 1ml Wine and beverages 8 ml 2 ml 30 min 4 5 ml 15ml 3 ml Feathers and plumes 2 units 1ml 60 min 1ml 0 8 ml 0 5 ml Raw meat 50 20 ml 30 min 1ml 0 8 ml 0 5 ml Blood 1ml 9ml 15 min 1ml 0 8 ml 0 9 ml Table 2 for these matrices a filtration step is not necessary t centrifuge between 6000 and 10000 x g for 20 minutes and remove the supernatant made of serum and fats leave the pellet only cut two feathers into 2 3 parts 1 5 cm beginning form the bulb 5 2 Add the amount of Solution A reported in Table 2 and then shake to homogenize the content 5 3 Incubate at 85 C 2 C following the time indications in Table 2 shake 2 3 times during the incubation period
6. iquot of supernatant Filtration Flour and corn seeds 5 09g 0 5 ml No Wheat flour 2 09 0 5 ml Yes Flour and soy seeds 0 59 1 0 ml Yes Generon SpyX products 5 09 0 5 ml No Seeds 2 50 0 5 ml Yes M ilk powder 5 09 0 5 ml Yes Soy proteins blood meat flour 2509 1 0 ml Yes Modified starch 2 59 2 5 ml No Lecithin granular and fluid 10 09 25ml No Glucose syrup and fruit juice 5 09 2 5 ml No Creams 5 09 2 5 ml No Chocolate creams coffee 5 0g 1 0 ml Yes Products soy based 5 0g 1 0 ml Yes Corn flakes and honey 5 0g 0 5 ml No Crackers grissinix bran polenta 5 0g 0 5 ml No Animal feed 250 0 5 ml Yes Starches and sugars 5 0g Zon No Raw and refined oils 20 09 2 5 ml No Pudding mustard jelly 5 09 2 5ml Yes Lyophilized products cheese 2 509 1 0 ml Yes Spices and dried vegetables 250 2 5 ml Yes Pizza 5 0g 1 0 ml Yes Sweets candy fruit 5 0g Zoi Yes Ready to use cake mixes 5 0g 2 5 ml Yes Jam 5 0g 1 0 ml Yes Tomato concentrate 5 0g 2 5 ml Yes Baby food 5 0g 2 5 ml Yes Vegetable matrices 2 59 0 5 ml No Flours and feed formulations 5 0g 0 5 ml Yes Cooked M eat fish and byproducts 5 0g 0 5 ml No Rice and derivatives 5 0g 1 0 ml Yes Margarine and butter 5 0g 2 5 ml Yes Vitamin and mineral supplements 5 0g 0 5 ml Yes Pet food yeast and ferments 5 0g 0 5 ml No Gastronomic specialities and Ice creams 5 0g 1 0 ml Yes Sauces and dips 5 0g 2 5 ml Yes Pasta 2 09 0 5 ml No Chips snacks and biscuits 5 09 2 5 ml Yes Gluten and sweet corn 250 1 0 ml N
7. o flavours 5 0g 2 5ml Yes Sorbitol and maltitol 5 09 2 5 ml No Nougat and dried fruit 500 1 0 ml Yes Table 1 suggested weights for sample extraction supernatant aliquots to be collected and filtration requirements for subsequent purification step Weights reported are only indicative for matrices with low DNA content it is possible to increase the starting quantity in order to obtain a more effective extraction for fluid and granular lecithin add to the sample 20 ml of n Hexane Shake gently and carefully avoid to turn the container upside down then add 10 ml of Solution A Again shake gently and leave it to rest for 10 minutes Proceed according to the step 4 2 of this protocol but the sample is processed at room temperature instead of 85 C User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15 5 4 DNA Extraction from Food and Feed 2 4 1 Add 20 ml of Solution A to the sample and then shake to homogenize the content 4 2 Incubate 1 hour at 85 C 2 C shake 2 3 times during the incubation period 4 3 Centrifuge the sample at speed between 6000 and 10000 x g for 10 minutes to convert g into rom see instruction manual of your own centrifuge 4 4 Transfer 1 ml of upper aqueous phase to a 2 ml tube For the matrices reported in RED in Table 1 it is suggested to split 3 ml of upper aqueous phase into 3 individual 2 ml tubes Note the upper phase is aqueous in samples containing fats or oils the aqueous phase lies in t
8. o a clean DNase RNase free 1 5 ml tube and add 150 ul of Solution D 100 ul for low DNA content matrices and Generon SpyX products 6 9 Wait 2 minutes then collect the column bound DNA by spinning the columns 30 seconds at 100 200 x g and continue at maximum speed for 5 minutes This DNA solution is stable one week at 4 C or 12 months at 20 C User guide lon Force DNA Extractor FAST Rev 32 of 06 08 15
9. trix see Table 1 filter the solution through a cellulose acetate membrane filter 0 45 um pore size inserted into a 10 ml syringe otherwise proceed directly to the next step 6 4 Connect DNA binding columns for DNA transfer to the vacuum box as indicated in Figure 1 Figure 1 connecting scheme of columns to the vacuum box A purification column supplied within ION Force extraction kit B Vacuum Box Adapter EXD010 A C syringe for ION Force ACC907 D Vacuum Box EXD010 P E column inlet 6 5 Set the vacuum pump to pressure between 0 5 and 0 9 atm alternatively verify that the flow rate is not faster than 1 drop second 6 6 Pour the content of every tube in the respective column and then proceed with columns wash by adding three times 0 75 ml of Buffer P previously reconstituted with absolute ethanol 6 7 Remove DNA binding columns from the vacuum box place each one in a collection tube and centrifuge between 4000 and 5000 g for 5 minutes to drain completely the Buffer P this step removes all ethanol traces which could inhibit the subsequent PCR reaction Note in order avoid Sample cross contaminations after each use it is important to clean up column inlets Figure 1 with 1 bleach sodium hypochlorite followed by abundant rinsing with distilled water The adapters for the Vacuum Box can be cleaned by keeping 1 hour in 1 bleach followed by abundant rinsing with distilled water 6 8 Transfer each column int
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