Contents Introduction
Contents
1. Before Starting Kit Contents Product No D3091 00 D3091 01 D3091 02 Purification times 5 Preps 50 Preps 200 Preps HiBind DNA Micro columns 5 50 200 2 ml Collection Tubes 15 150 600 Buffer ACL 5 ml 50 ml 200 ml Buffer ACB 10 ml 100ml 2x200ml Buffer ACW1 5 ml 50 ml 150 ml Carrier RNA 310 ug 310 ug 2x1mg DNA Wash Buffer 5 ml 20 ml 3x 20 ml Elution Buffer 5 ml 40 ml 160 ml OB Protease 10 mg 100 mg 4 x 100mg Protease Storage Buffer 1 ml 6 ml 24 ml User Manual 1 1 1 IMPORTANT 1 Reconstitute OB Protease in 500 ul 5 preps 5 ml 50preps or 4 x 5 ml 200 preps Protease Storage Buffer Vortex vial briefly prior to use We recommend that you aliquot and store vials of reconstituted protease at 20 C 2 DNA Wash Buffer Concentrate must be diluted with absolute ethanol 96 100 as follows D3091 00 Add 20 ml absolute ethanol D3091 01 Add 80 ml absolute ethanol D3091 02 Add 80 ml absolute ethanol per bottle Store diluted DNA Wash Buffer at room temperature All centrifugation steps must be carried out at room temperature Page 3 of 8 Introduction E Z N A Circulating DNA Kit provides a rapid and easy method for the isolation of Circulating DNA from plasma serum and other cell free body fluids Samples can be either fresh or frozen provided that they have not been frozen and thawed more than once The kit allows single or multiple simultaneous processing of samples in under 120 minutes There is no need for ph2. Binding Capacity Each HiBind column can bind approximately 20 ug DNA Page 2 of 8 Determination of Yield and Quality The total DNA yield can be determined by a spectrophotometer using deionized water Tris HCl buffer or Elution Buffer as blank Dilute the DNA in TE buffer and calculate concentration as DNA Absorbance x 0 05 ug ul x Dilution factor The quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm A ratio of Aj o Ays9 Of 1 7 1 9 corresponds to 85 95 purity Troubleshooting Guide Problem Possible Cause Suggestions Clogged Column Add the correct volume of Buffer BL Incomplete lysis and incubate for specified time at 70 C It may be necessary to extend incubation time by 10 min Sample too viscous Divide sample into multiple tubes adjust volume to 250 Ul with 10 mM Tris HCl Poor elution Repeat elution or increase elution Low DNA yield volume see note on page 4 Incubation of column at 70 C for 5 min with Elution Buffer may increase yields Improper washing Wash Buffer Concentrate must be diluted with absolute 100 ethanol as specified on page 5 before use Page 7 of 8 A Spin Protocol Purification of Circulating DNA from 0 1 1ml Plasma or Serum Materials and equipments Supplied by User Tabletop microcentrifuge and sterile 1 5 ml tubes Water bath set to 65 C Ethanol approximately 0 3 ml per sample RNas
3. Problem Low A ratio A 260 280 No DNA eluted Washing leaves colored residue in column Page 8 of 8 Possible Cause Extended centrifugation during elution step Hemoglobin remains on column Poor cell lysis due to improper mixing with Buffer ACL No ethanol added to Wash Buffer Concentrate Incomplete lysis due to improper mixing with Buffer ACL No ethanol added to Wash Buffer Concentrate Suggestions Resin from the column may be present in eluate Avoid centrifugation at speeds higher than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digests After application of sample to column wash once with 300 ul Buffer AL Mix thoroughly with Buffer BL prior to loading HiBind column Dilute Wash Buffer with the indicated volume of absolute ethanol before use Buffer BL is viscous and the sample must be mixed thoroughly Dilute Wash Buffer with the indicated volume of absolute ethanol before use Contents Introduction Storage and Stability Binding Capacity Kit Contents Before Starting A Circulating DNA Spin Protocol B Ciuculating DNA Vacuum Protocol Determination of Yield and Quality Troubleshooting Guide B Vacuum Protocol Purification of Circulating DNA from Plasma or Serum Material and equipment
4. e A Prepare a stock solution of RNase A at 50 mg ml NOTE The procedure below has been optimized for use with FRESH or FROZEN Plasma or Serum samples from0 1 to 1 ml in volume Other Cell free samples can also be used For DNA extraction from Blood we suggest using the E Z N A Blood DNA Kit product number D3392 To isolate Circulating RNA from serum or other non cellular body fluids use E Z N A Circulating RNA Kit Preheat an aliquot of Elution Buffer approximately 0 1 ml per sample at 65 C Carry out all centrifugation steps at room temperature Add m25 ul OB Protease 50 ul OB Protease or 100 ul OB Protease to a sterile microcentrifuge tube Add 250 ul 500 ul or 1ml plasma or Serum to the tube containing protease Add m 200 ul ACL Buffer and 5 6 ul Carrier RNA 400 ul ACL Buffer and 5 6 ul Carrier RNA or 0 8ml Buffer ACL and 5 6 ul Carrier RNA Vortex at maxi speed for 30s to mix thoroughly Incubate at 600C for 30 min Add m 450 ul ACB Buffer 900 ul ACB Buffer or 1 8ml Buffer ACB Vortex at maxi speed for 30s to mix thoroughly Incubate the mixture on ice for 10 min Assemble an HiBind DNA Micro column in a 2 ml collection tube provided Transfer 750 ul of the lysate from step 6 into the column Centrifuge at 8 000 x g for 1 min to bind DNA Discard flow through liquid and assembe the column into the same collection tube Page 4of 8 10 11 12 13 14 15 Repeat step 8
5. eluted DNA at 20 C Page 5of 8
6. enol chloroform extractions and time consuming steps such as CsCl gradient ultracentrifugation and precipitation with isopropanol or ethanol are eliminated DNA purified using the E Z N A Circulating DNA method is ready for applications such as PCR Circulating detection and genotyping E Z N A Circulating DNA Kit uses the reversible nucleic acid binding properties of HiBind matrix combined with the speed of mini column spin technology A specifically formulated buffer system allows Circulating DNA bind to the matrix Samples are first lysed under denaturing conditions and then applied to the HiBind DNA spin columns to which DNA binds while cellular debris hemoglobin and other proteins are effectively washed away High quality DNA is finally eluted in sterile deionized water or low salt buffer Storage and Stability All components of the E Z N A Circulating DNA Kit except the OB Protease should be stored at 22 C 25 C Once reconstituted in water OB Protease must be stored at 20 C Under these conditions DNA has successfully been purified and used for PCR after 24 months of storage Under cool ambient conditions a precipitate may form in the Buffer ACL and ACB In case of such an event heat the bottle at 37 C to dissolve Store Buffer ACL and ACB at room temperature Expiration Date All E Z N A Circulating DNA Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22 25 C
7. s supplied by user Tabletop microcentrifuge and sterile 1 5 ml tubes Vacuum Manifold Water bath set to 65 C Ethanol approximately 0 3 ml per sample RNase A Prepare a stock solution of RNase A at 50mg ml Prepare the lysate by following step 1 6 of Protocol A Spin protocol on page 4 Insert the HiBind DNA Micro column into the vacuum manifold Carefully apply the lysate to an HiBind DNA column Turn on the vacuum source to draw all liquid through the column when all lysates have been drawn through the column completely switch off the vacuum pump Note If the lysate has difficulty to pass through the column at this stage Place the column into a collection tube supplied Close the lid and centrifuge at 8000 x g for 5 minutes or until all liquid pass through the column Place the column into another collection tube supplied and continue step 7 of the spin protocol Pipet 700 ul of Buffer ACW1 into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Wash the column by pipetting 700 ul of DNA Wash Buffer diluted with ethanol into the column Turn on the vacuum source to draw all liquid through the column Turn off the vacuum Close the lid of HiBind DNA column remove it from the vacuum manifold Insert the column into a collection tube supplied and centrifuge at 15 000 x g for 2 minute to completely dry the column Elute DNA as Step 13 15 on page 5 Page 6 of 8
8. until all of the lysate pass through the Micro column Place the column into a second 2 ml tube provided and wash by pipetting 700ul of Buffer ACW1 Centrifuge at 8 000 x g for 1 min Again Discard flow through liquid and reuse the collection tube for next step Place the column into a same 2 ml tube from step 10 and wash by pipetting 700 ul of DNA Wash Buffer diluted with ethanol Centrifuge at 8 000 x g for 1 min Again dispose of collection tube and flow through liquid Note that DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle or page 3 If refrigerated the diluted wash buffer must be brought to room temperature before use Using a new collection tube wash the column with a second 700 ul of DNA Wash Buffer and centrifuge as above Discard flow through and re use the collection tube for next step Place the empty column into the same 2 ml collection tube form step 12 centrifuge at maximum speed 13 000 x g for 2 min to dry the column This step is crucial for ensuring optimal elution in the following step Place the column into a sterile 1 5 ml microfuge tube and add 20 50 ul of preheated 65 C Elution Buffer Allow tubes to sit for 5 min at room temperature To elute DNA from the column centrifuge at 8 000 x g for 1 min Retain flow through containing the DNA Place column into a second 1 5 ml tube Elute DNA again as step 11 12 Discard column and store the
Download Pdf Manuals
Related Search