Contents Introduction Principle Storage and Stability
Contents
1. Incubate at room temperature for 10 minutes Note see the instruction for preparing Carrier RNA on page 4 3 Add 800ul isopropanol to each sample mix throughly by pipetting or vortexing Incubate 3 5minutes at room temperature 4 Place the centrifuge Tube on a magnetic separation device to magnetize the magnetic particles Leave the tube on the magnet until all the magnetic particles are pelleted The capture time is depend on the sample type and magnetic stand used 5 Carefully aspirate and discard the supernatant without disturbing the magnetic particles 6 Remove the Processing Tube containing the magnetic particles from the magnetic separation device Add 400 ul of Buffer MBW and resuspend magnetic particles pellet by pipetting up and down 20 times or vortex the tube at maximum speed for 1 minute 7 Place the tube onto a magnetic separation device to magnetize the magnetic particles Aspirate and discard the cleared supernatant after the lysate is cleared 8 Remove the tube containing the magnetic particles from the magnetic separation device Add 500 ul of SPR Wash Buffer to each well and resuspend magnetic particles pellet by pipetting up and down or vortexing for 30 seconds 9 Place the tube onto a magnetic separation device to magnetize the magnetic particles Aspirate an discard the cleared supernatant after the lysate is cleared 10 11 12 13 14 15 Add another 500 ul of SPR Wash Buffer and resusp2. Contents Introduction eriei hele ee Ba AG ete eed et on ne E 2 Pinciples set 48 30 pags heck Re odes eit ada be Dees Bde See a itil 2 Storage and Stability iee a e cee 2 Kit Contents icy fas ia a gil wade we O eae A Hey E Boe elk 3 Before St rting rer seine Si Me eet he ig T he ihe pk ee oe ead 3 Mag Bind Viral DNA RNA Protocol 200 pl 0 200 002 eee 4 Mag Bind Viral DNA RNA Protocol 400 pl 20200 02 eee eee 6 Troubleshooting Guide 0 0000 cee 8 Revised September 2008 Introduction The E Z N A Mag Bind Viral DNA RNA Kit is designed for rapid and reliable isolation of viral DNA and RNA from serum plasma and other cell free body fluids The Mag Bind Magnetic Beads technology provide high quality RNA and RNA which is suitable for direct use in most downstream applications such as amplifications and enzymatic reactions This system can be easily adapted with an automated system and the procedure can be scaled up or down Principle If using the Mag Bind Viral DNA RNA Kit for the first time please read this booklet to become familiar with the procedure and its various modifications Samples are lysed in a specially formulated buffer containing detergent and chaotropic salt After adjust the buffer condition nucleic acids DNA RNA will form a complex with magnetic beads The beads nucleic acids complexis separated from lysates using a magnet Proteins and cellular debris are efficient
3. ate an discard the cleared supernatant after the lysate is cleared Add another 400 ul of SPR Wash Buffer and resuspend magnetic particles pellet by pipetting up and down for 20 times or vortexing at maximum speed for 30 seconds Place the tube onto a magnetic separation device to magnetize the Mag Bind particles Aspirate and discard the cleared supernatant Carefully remove any liquid drop from tube Leave the tube on the magnet stand Air dry the magnetic particles at room temperature for 7 10 minutes Add 20 50 pl DEPC Treated water and resuspend magnetic particles pellet by vortexing or pipetting up and down for 20 times Incubate at room temperature for 3 minutes Place the tube onto a magnetic separation device to magnetize the Mag Bind particles Transfer the cleared supernatant contains purified DNA and RNA into a new RNase free microtube Mag Bind Viral DNA RNA Isolation Protocol for 400ul serum or plasma The following protocol is designed for isolating viral DNA and RNA from 400 ul cell free body fluids such as plasma or serum This protocol should use 2 0 mL centrifuge tube and suitable magnetic separation device 1 Add 400 ul sample serum plasma or other cell free body fluids into appropriate Processing Tube not supplied followed by adding 10 ul Proteinase K solution and 10 ul Mag Bind Particle RV 2 Add 400 pl MVL Carrier RNA Buffer mix to the sample Mix throughly by pipetting or vortexing
4. eads make sure the lysate is clear of particles before adding magnetic beads Do not over dry the magnetic beads No RNA eluted SPR and MBW Wash Buffer Concentrate not diluted with absolute ethanol and isopropanol Prepare MBW and SPR Wash Buffer Concentrate as instructed on the label Problem with Insufficient RNA 1 RNA in the sample is already downstream was used degraded do not freeze and thaw application the sample more than once or store at room temperature for too long 2 Quantify the purified DNA RNA accurately and use sufficient elute Carryover of the magnetic beads in the elution Carryover from the magnetic beads in To remove the carryover magnetic beads from eluted DNA RNA simply the eluted RNA will magnetize the magnetic beads and not effect carefully transfer to a new tube or downstream tube applications
5. end magnetic particles pellet by pipetting up and down for 20 times or vortexing at maximum speed for 30 seconds Place the tube onto a magnetic separation device to magnetize the Mag Bind particles Aspirate and discard the cleared supernatant Carefully remove any liquid drop from tube Leave the tube on the magnet stand Air dry the magnetic particles at room temperature for 7 10 minutes Add 50 100 pl DEPC Treated water and resuspend magnetic particles pellet by vortexing or pipetting up and down for 20 times Incubate at room temperature for 3 minutes Place the tube onto a magnetic separation device to magnetize the Mag Bind particles Transfer the cleared supernatant contains purified DNA and RNA into a new RNase free microtube Trouble Shooting Problem Likely Cause Low RNA yields Suggestions i Resuspend the magnetic particles by vortexing before use Incomplete resuspension of magnetic particles RNA degraded during sample storage Make sure the sample is properly stored and make sure the samples are processed immediately after collection or removal from storage SPR Buffer were not prepared correctly Prepare the SPR Buffer by adding ethanol according to instruction Be careful not to remove the Loss of magnetic magnetic beads during the operation beads during operation Undissolved particles in the cell lysate cause congregation of magnetic b
6. ly washed away by a washing step Purified DNA and RNA are then eluted in nuclease free water or low ionic strength buffer DNA and RNA purified with this method can be directly used in most downstream applications without the need for further purification Storage and Stability Mag Bind Viral DNA RNA Kit are stable for at least 12 months from date of purchase During shipment or storage in cool ambient conditions precipitates may form in the MVL Buffer Mag Bind Particle R need to be stored at 4 8 C Kit Contents m During the procedure work carefully but quickly Product M6245 00 M6245 01 M6245 02 7 Under cool ambient conditions crystals may form in MBW Buffer This is normal and Purification 5 50 200 the bottle should be warmed to re dissolve the salt Mag Bind Particles R 100 uL 2 mL 8 mL 7 Prepare Carrier RNA Dissolve Carrier RNA with DEPC Water to the final concentration at 1mg ml Dissolve the dissolved Carrier RNA into convenient aliquot Buller MVE 1ml 2X 30m 2x200 ML and store at 20 C Add 3 ul Carrier RNA for each milliliter of MVL Buffer before Proteinase K 3 mg 60 mg 240 mg use Buffer MBW 6 ml 100 ml 400 ml SPR Wash Buffer 25 mL 12 mL 50 mL Material and Equipments supplied by User DEPC Water 10 mL 100 mL 400 mL Have the following reagents and supplies ready before starting procedure 7 Magnetic separation device for 1 5 OR 2 0 mL centrifuge tubes Carrier RNA 50 pg 320 ug 1600 yg 1 5 mL or 2 0 Ce
7. ntrifuge Tubes m Absolute ethanol 96 100 m Isopropanol m Ethanol 96 100 User Manual 1 1 1 MVL Contains guanidine salts Do not use bleach as disinfectant Before Starting Mag Bind Viral DNA RNA Isolation Protocol for 200ul serum or SPR Wash Buffer must be diluted with absolute ethanol before use plasma M6245 00 Add 10 mL 100 ethanol M6245 01 Add 48 mL 100 ethanol The following protocol is designed for isolating viral DNA and as from 200 ul cell free body fluids such as plasma or serum The protocol can be easily scale up to process up M6245 02 Add 200 mL 100 ethanol to 400 ul sample by using appropriate processing tube and suitable magnetic separation IMPORTANT device Dissolve Proteinase K as following and store at 20 C M6245 00 Add 75 ul DEPC Water 1 Add 200 ul sample serum plasma or other cell free body fluids into a 1 5 mL Centrifuge Tube followed by adding 10 ul Proteinase K solution and 10 ul Mag Bind Particle RV M6245 01 Add 750 ul DEPC Water M6245 02 Add 3 2 ml DEPC Water Please take a few minutes to read this booklet thoroughly and become familiar with i 2 Add 200 pl MVL Carrier RNA Buffer mix to the sample Mix throughly by pipetting or vortexing Incubate at room temperature for 10 minutes the protocol Prepare all materials required before starting in order to minimize d Note see the instruction for preparing Carrier RNA on page 4 RNA degradation Wear gloves pr
8. otective goggles and take great care when working with chemicals 3 Add 400ul isopropanol to each sample mix throughly by pipetting or vortexing Whenever working with RNA always wear latex gloves to minimize RNase Incubate 3 5minutes at room temperature contamination Use only clean RNase free disposable plastic pipette tips when using the supplied reagents 4 Place the Centrifuge Tube on a magnetic separation device to magnetize the 10 11 12 13 14 15 magnetic particles Leave the tube on the magnet until all the magnetic particles are pelleted The capture time is depend on the sample type and magnetic stand used Carefully aspirate and discard the supernatant without disturbing the magnetic particles Remove the centrifuge tube containing the magnetic particles from the magnetic separation device Add 300 ul of Buffer MBW and resuspend magnetic particles pellet by pipetting up and down 20 times or vortex the tube at maximum speed for 30 seconds Place the tube onto a magnetic separation device to magnetize the magnetic particles Aspirate and discard the cleared supernatant after the lysate is cleared Remove the tube containing the magnetic particles from the magnetic separation device Add 400 ul of SPR Wash Buffer to each well and resuspend magnetic particles pellet by pipetting up and down or vortexing for 30 seconds Place the tube onto a magnetic separation device to magnetize the magnetic particles Aspir
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