Contents - Biomiga
Contents
1. 01 or 216 mL R6619 02 10090 ethanol to RNA Wash Buffer before use The final concentration of 100 ethanol should be 80 During shipment or storage under room temperature crystal may form in certain buffers Dissolve the precipitates at 37 C before use Do not freeze the buffers at any time Preheat the Buffer RB and DEPC treated ddH O 100 uL per sample at 65 C Optional Add 600 uL R6619 00 or 7 2 mL R6619 01 or 36 mL R6619 02 10096 ethanol to DNase Stop Buffer before use Smaples fresh tissue is preferred for RNA integrity Materials supplied by users v v v v Page 4 Tabletop microcentrifuge 10096 ethanol p mercaptoethanol for denaturing the endogenous RNase Chloroform and isoamyl alcohol 24 1 for removal of polysaccharides and proteins Optional DNase I EZgene Mollusk RNA Mini Kit Avoiding RNase Contamination Please prepare materials as following instructions when working with RNA l For RNA use only Keep a separate set of pipettors for RNA use to avoid contamination with RNases Avoid touching the barrel or metal ejector to the sides of tubes Solutions Store solutions in small aliguots and discard each aliguot after use Electrophoresis apparatus Wash with detergent solution rinse in H 0 and dry with ethanol Then fill with 3 solution of H O Don t use DEPC solutions because it will break down the plastic incubate 10 mins at room temperature and rinse wi2. All other components can be stored at room temperature 22 25 C All kit components are guaranteed for 1 year from the date of purchasing Page 2 EZgene Mollusk RNA Mini Kit Kit Contents Catalog R6619 00 R6619 01 R6619 02 Preps 4 50 250 ezBind Columns 4 50 250 Collection Tubes 8 100 500 1 5 mL RNase free microfuge tube i a nd Buffer BML 2ml 20 mL 100 mL Buffer LY 4 mL 30 mL 130 mL Buffer RB 4 mL 45 mL 220 mL RNA Wash Buffer 2mL 20 mL 54 mL DEPC Treated ddH O 500 uL 20 mL 70 mL DNase I Stop Buffer 400 uL 4 8 mL 24 mL User Manual 1 1 1 DNase I and RNase Inhibitor not supplied They could be purchased from Biomiga Safety Information Buffer LY contains chaotropic salts which may form reactive compounds when combines with bleach Do not add bleach or acidic solutions directly to the preparation waste ware gloves and protective eyewear when handling Page 3 EZgene Mollusk RNA Mini Kit Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps Important v Determine amount of Buffer BML and Buffer LY to be used add 20 uL B mercaptoethanol B ME per 1 mL Buffer BML or LY Buffer BML and Buffer LY contains B ME can be stored at room temperature for up to 1 month B ME is key in denaturing endogenous RNase Add 8 mL R6619 00 or 20 mL R6619
3. for up to 24 hours at 4 C for up to a week and 20 C or 70 C for long term RNA guality It is highly recommended that RNA guality be determined before downstream applications The guality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium bromide staining Several sharp bands should appear on the gel including 28S and 18S ribosomal RNA bands as well as certain populations of mRNA and bands If these bands smear towards lower molecular weight RNAs then the RNA has undergone major degradation during preparation handling or storage RNA molecule less than 200 bases in length do not efficiently bind to the RNA column An A26 A2s0 ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Page 6 EZgene Mollusk RNA Mini Kit Protocol for Total RNA Extraction From Mollusks Invertebrates II Grind no more than 30 mg tissue in liquid nitrogen with mortar and pestle and transfer the powder into a clean 1 5 ml microcentrifuge tube Note The procedure could be scaled up with the increase of starting material and volumes of all buffers With RNA binding capacity of 100 ug tissue more than 50 mg is not suggested Less tissue or more buffer is suggested for hard to lysis tissue Quick protocol follow step 2 to 4 and proceed to step 10 Add 350 uL Buffer LY Add fi ME before use and vortex vigorously to make sure that all clumps are dispersed Note Complete dispersion is critical for RNA
4. increase the yield while lower the Note For higher RNA concentration reload the first eluate for the second elution Determine the concentration and purity of RNA measure absorbance at 260 nm and 280 nm in a spectrophotometer A ratio of 1 8 2 0 corresponds to 90 100 pure nucleic acid Store RNA samples at 70 C in water Page 9 EZgene Mollusk RNA Mini Kit Trouble Shooting Guide Problems Possible reasons Suggested Improvements Low Aogo Aogoratios Protein contamination Do a Phenol Chloroform extraction Loss of total RNA up to 4096 should be expected Low Aogco Aogoratios Guanidine Thiocyanate Add 2 5 volumes of ethanol and 0 1M contamination NaCl final concentration to precipitate RNA Incubate for 30 min at 20 C Centrifuge at 10 000 g for 15 min at 47C Resuspend the RNA pellet in DEPC treated water Low Yield RNA in sample Freeze samples immediately in liquid degraded nitrogen and store at 70 C after collect it Low yield RNA remains in the Pre heat DEPC treated ddH O and column repeat elution Low Yield The binding capacity of Use of too much tissue sample the membrane in the exceeding the binding capacity of spin spin column was column will cause the decreasing of exceeded total RNA yield Low Yield Ethanol not added to Add ethanol to the RNA Wash Buffer buffer and DNase Stop Solution before purification Genomic DNA Too much total RNA Reduce total R
5. Contents Introd ction nic n d eter ettet iode ite a i 2 Storage and Stability reete eter 2 Kit Contents uere et ene e E ER ERU IER Ua 3 Before Starting ace e ato diete e ter Ree Re SS 1 Protocol For Extracting Total RNA From Mollusc 7 TroubleShootingGuide 10 Related Products eere tete ES N ES 11 LimiteedUseandWaranty 12 Page 1 EZgene Mollusk RNA Mini Kit Introduction The EZgene Mollusks RNA maxi Kit is designed for efficient recovery of total RNA greater than 200 nt from molluscs roundworms flatworms and other invertebrate tissue samples rich in mucopolysaccharides The procedure relies on the well established properties of the cationic detergent cetyltrimethyl ammonium bromide CTAB in conjunction with the selective RNA binding of silica membrane Samples are homogenized and lysed in a high salt buffer containing CTAB and extracted with chloroform to remove mucopolysaccharides and denature proteins Following a rapid alcohol precipitation step binding conditions are adjusted and RNA further purified using ezBind RNA spin columns In this way salts proteins and other contaminants are removed to yield high quality total RNA suitable for downstream applications such as reverse transcription poly A mRNA selection and hybridization techniques Storage and Stability DNase I optional should be stored at 20 C
6. NA amount used in RT contamination sample was used in RT PCR to 50 100 ng PCR Genomic DNA The sample may contain Reduce the amount of starting tissue in contamination too much genomic the preparation of the homogenate DNA Most tissues will not show a genomic DNA contamination problem at 30 mg or less per prep Increase buffer volume and do multiple loadings to column Page 10 EZgene Mollusk RNA Mini Kit Related EZgene Products Catalog Product Name Preps Price R6311 02 25 650 00 R6411 02 25 680 00 R6812 02 96 well blood RNA kit 20x96 3500 00 leleleolgle lel lelgle Page 11 EZgene Mollusk RNA Mini Kit Limited Use and Warranty This product is warranted to perform as described in its labeling and in Biomiga s literature when used in accordance with instructions No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Biomiga Biomiga s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of Biomiga to replace the products Biomiga shall have no liability for any direct indirect consequential or incidental damage arising out of the use the results of use or the inability to use it product For technology support or learn more product information please visit our website at www biomiga com or conta
7. ct us at 858 603 3219 Page 12 EZgene Mollusk RNA Mini Kit
8. extraction Centrifuge 10 000 x g for 2 min at room temperature Carefully transfer the supernatant to a clean 1 5 ml microfuge tube Avoid the milky interface containing contaminants and inhibitors Add 350 uL 10046 ethanol and mix by sharp hand shaking Suggested but not mandatory protocol follow step 5 to 9 and proceed to step 10 Add 350 uL Buffer BML Add f ME before use and vortex vigorously to make sure that all clumps are dispersed Note Complete dispersion is critical for RNA extraction Add 350 uL chloroform isoamyl alcohol 24 1 and vortex to mix Centrifuge 10 000 x g for 2 min at room temperature Carefully transfer Page 7 EZgene Mollusk RNA Mini Kit 10 11 12 the supernatant to a clean 1 5 ml microfuge tube Avoid the milky interface containing contaminants and inhibitors Note This step will remove much of the polysaccharides and proteins from solution and improve the downstream application Add 1 volume of isopropanol and mix to precipitate RNA Immediately centrifuge 10 000 x g for 2 min at room temperature Remove the supernatant completed Avoiding disturbing RNA pellet Invert the microfuge tube on a paper towel for 1 min to allow residual liquid to drain Add 100 uL of Buffer LY Add f ME before use and pre heated to 65 C and vortex to resuspend the pellet Note Pre heating the buffer to 65 C is necessary to effectively dissolve the RNA Add 350 uL Buffer LY Add f ME before
9. th DEPC treated H5O Glassware Bake glassware at 300 C for 4 hours or 1809C or higher for several hours Alternatively soak glassware in freshly prepared 0 196 v v DEPC in water or ethanol for 1 hour drain and autoclave I t is necessary to destroy any unreacted DEPC which can otherwise react with other proteins and RNA Plasticware Treat plasticware with DEPC Use RNase free disposable tips and tubes Use sterile forceps to transfer items to racks Gloves Use gloves from a fresh box at all times Don t touch the gloves to any surface that might be contaminated with RNases Work carefully and quickly during the procedure Page 5 EZgene Mollusk RNA Mini Kit Removal of genomic DNA using DNase digestion DNA digestion is necessary for downstream applications that are sensitive to very small amounts of DNA for example RT PCR with low abundance target Generally it is not required to do so since the EZgene RNA purification kit selectively isolates RNA and eliminates most of the DNA If there is DNA contamination either reduces the tissue amount or cell Stabilization of RNA in harvested animal tissues The intact of RNA in harvested tissue will be protected with the addition of RNASecure solution Biomiga catalog R1011 1 Cut the tissue into slices less than 0 5cm thick and immediately add at least 15 volumes of RNAsecure solution for example 150 uL RNAsecure solution per 10 mg tissue 2 Store at room temperature
10. use and 250 pL 100 ethanol and mix by sharp hand shaking Immediately apply the entire mixture including any precipitation to the DNA column with collection tube Centrifuge 10 000 x g for 15 sec at room temperature to let the solution pass through the column Discard the flow through liquid and reinsert the column into the collection tube Optional Add 50 uL DNase I 2U RNase free solution onto the middle of the column and incubate at room temperature for 15 min Add 200 uL DNase Stop Buffer onto the column and centrifuge at 14 000 rpm for 1 min Discard the flow through Add 500 uL Buffer RB and centrifuge at 10 000 x g for 15 sec Discard the flow through liquid and collecting tube Page 8 EZgene Mollusk RNA Mini Kit 13 14 15 16 17 Place the column in a clean 2 mL collection tube and add 500 uL RNA Wash Buffer Add ethanol before use Centrifuge at 10 000 x g for 15 sec at room temperature and discard flow through and reinsert the column into the collection tube Repeat this step Centrifuge the DNA column for 2 min at full speed to completely dry the ethanol remain on the membrane Note Complete removal of residual ethanol is critical for RNA elution Transfer the column to a clean 1 5 ml microfuge tube Add 50 100 uL of DEPC Treated ddH5O to the center of the membrane to elute the RNA Centrifuge 1 min at maximum speed 80 RNA is recovered with first elution Optional A second elution will
Download Pdf Manuals
Related Search