Contents - Biomiga
Contents
1. most micro centrifuges Discard flow through liquid from previous step Apply the remaining sample and centrifuge as above Again discard flow through liquid and reuse 2 mL collecting tube in next step Wash the column by adding 700 ul RNA Wash Buffer Add absolute ethanol before use Centrifuge as above Discard liquid and 2 m L collecting tube Place column into a New 2 mL collection tube Pipet 500 ul RNA Wash Buffer onto spin column Centrifuge as above and discard flow through and re use the collection tube in next step Place the column into the same collection tube from previous step Spin empty column 2 m in at 10 000 x g to dry ezBind matrix This is critical for removing residual ethanol which m ay otherwise be carried over during elution and interfere with downstream applications Transfer spin column to a new 1 5 m L microfugetube Elute the probe by pipetting 100 ul DEPC treated water supplied directly onto m atrix Centrifuge 1 minute at 8 000 x g approx 10 000 rpm on most micro centrifuges For non isotopic probes usually present in microgram quantities repeat the elution with a second 100 ul aliquot of Page 7 EZgene Poly Gel RNA Purification Kit DEPC water to improve yield Probes lt 250 nt An optional phenol chloroformextraction followed by precipitation with ethanol is suggested for RNA molecules lt 250 nt For ethanol precipitation add ammonium acetate to a final concentration of 0 5 M and add2. 2 5 x volice cold ethanol Radioisotopic probes may also require 10 ug of yeast tRNA as carrier Non isotopic probes will usually not need carrier as higher amounts are eluted from the gel Note that phenol extractions should not be performed with digoxigenin labeled probes since RNA will separate into the organic phase III Probe Quantitation Radioisotopic Probes Analyze the eluted probe by scintillation counting to determine the concentration cpm mL A typical transcription reaction should yield approximately 2 6 x 104 cpm ul in 100 ul Probe should be stored at 20 C or 70 C for greater stability Non isotopic Probes Measure the OD readings at 260 and 280 nm The 260 280 ratio should be 1 9 2 0 Low 260 280 ratios indicate protein acrylam idecontamination which can lead to inaccurate concentration calculations Based on OD260 reading and the conversion 1 A2 unit 40 ug RNA calculate the concentration of the probe A typical 20 uL transcription reaction containing 0 5 m M of each NT P and subsequent gel purification should yield about 4 8 ug of R N A The probe should be stored at 20 C or 70 C for greater stability Degradation typically starts after 5 10 freeze thaw cycles so the probe should be stored as aliquots Page 8 EZgene Poly Gel RNA Purification Kit
3. 500 Buffer CS 2mL 20 mL 100 mL Buffer LY 4 mL 50 mL 250 mL RNA Wash Buffer 4 mL 24 mL 80 mL DEPC treated H O 1 2 mL 20 mL 100 mL User Manual 1 1 1 Page 2 EZgene Poly Gel RNA Purification Kit Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps Important e Add 16 mL RC3612 00 or 96 mL RC3612 01 or 320 mL RC3612 02 96 100 ethanol to RNA Wash Buffer before use e Buffer may form crystals under cool ambient conditions It is critical to warm up the buffers at 37 C to redissolve the salt e Add 20 uL of B mercaptoethanol B mercaptoethanol per 1 mL of Buffer RB before use This mixture can be stored at room temperature up to 1 month e All centrifugation steps must be carried out at room temperature Materials supplied by users e 96 100 ethanol e Micro centrifuge capable of 10 000 x g e RNase free microcentrifuge tubes and tips e Disposable latex gloves Page 3 EZgene Poly Gel RNA Purification Kit Avoiding RNase Contamination Please prepare materials as following instructions when working with RNA 1 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 aliquots and discard each aliquot after use Electrophoresis apparatus Wash with detergen
4. Contents TMEOGUC HOM ieee fees as lesteveen ka cece Uecsececnedeaeads ondeasantaee testes dxctadeeecadentes 2 Storage and Stability 0 ccc cece cee ene ee eee eee eee eee e ences 2 Kit COMLCMIS ss sive st ceed ottaa aE Habe Gals Cat eck E ETES aces eke ott AE ETES 2 Before Starting vives a scadcossrins a oaiecereperele n o Salen a Soule 3 Avoid Rnase Contamination e cece eee eeeeeeee ene eneeneenens 4 Polyacrylamide Gel Recovery Protocol eceeeeeeeee ene 5 Page 1 EZgene Poly Gel RNA Purification Kit Introduction The EZgene Poly Gel RNA Recovery Kit provides an easy and reliable method for recovering RNA probes from denaturing acrylamide gels The probe is eluted by passive diffusion from the gel fragment with a unique buffer system and further purified using an ezBind RNA spin column The procedure can produce enough probes ready for hybridization in 1 4 hours The eluted RNA is ultra clean and ready for subsequent analysis and molecular manipulation Storage and Stability All components of the Poly Gel RNA Recovery Kit are stable for at least 24 months from the date of purchase when stored at 22 25 C Once diluted RNA Wash Buffer Concentrate must not be refrigerated Kit Contents Catalog RC3612 RC3612 01 RC3612 02 00 Preps 4 50 250 RNA columns 4 50 250 Filter Units 4 50 250 Collection Tubes 8 100
5. bridization reaction This allows setup of hybridization reactions on the same day as probe preparation Note that elution of a high specific activity probe in 200 uL of elution buffer should yield approximately 2 5 x 10 cpm uL II RNA Probe Clean Up All subsequent steps are required only if downstream application involve enzymatic reactions such as reverse transcription Carry out these steps carefully but quickly Probes gt 250 nt Transfer gel and buffer to a Poly Gel filter unit mounted in a sterile 1 5 mL microcentrifuge tube Use a blue pipette tip with the end cut to do this Centrifuge at 10 000 x g for 5 min at room temperature to filter the sample To the eluate add 800 uL Buffer RB B mercaptoethanol B ME and vortex briefly to mix With radioisotopic probes also add 5 ug yeast tRNA in no more than 25 uL as carrier Add 600 uL absolute ethanol and immediately vortex for 1 min A precipitate may form on addition Page 6 EZgene Poly Gel RNA Purification Kit 10 11 of ethanol This will not interfere with the procedure and should be thoroughly resuspended Proceed to the next step without delay Note Remember to add 20 uL 2 mercaptoethanol per 1 mL of Buffer RB before use This mixture is stable at room temperature for 1 week Apply 700 pL of sample from previous step onto ezBind RNA spin column Red assembled in a 2 mL collection tube supplied Centrifuge 20 seconds at 10 000 x g approx 12 500 rpm on
6. nd on the TLC plate or band in the gel The smaller the size of this gel fragment the better the elution efficiency This band should be the most intense band present If you are concerned that not all the probe was cut out visualize the gel again with UV light to verify that the probe band is gone 3 Transfer the gel fragment onto a nuclease free microscope slide Mash and pulp the gel completely with a second glass slide or nuclease free razor Carefully transfer gel pulp to a nuclease free microcentrifuge tube and add 200 uL Buffer PGC A slice of 2 mm x 5 mm x 0 75 mm corresponds to 200 uL Note For a larger fragment increase the volume of Buffer PG until the gel is covered 4 Incubate the tube at 65 C for 1 2 h Note The elution time depends on the size of the gel fragment RNA Page 5 EZgene Poly Gel RNA Purification Kit size and the temperature of the incubation About 90 of a 400 nt transcript could be eluted in 1 hr at 65 C Larger fragments will take longer to elute Proceed to step 3 for RNA clean up if downstream applications involve enzymatic manipulation Note It is not necessary to elute all the probe prior to hybridization only what will be needed i e 5 10 x 10 cpm of a high specific activity probe per RNASE protection assay reaction or 1x 10 cpm for Northern Assays An aliquot of Buffer PGC containing some of the probe can be removed at any time during the elution and used directly in the hy
7. t solution rinse in H 0 and dry with ethanol Then fill with 3 solution of H20 Don t use DEPC solutions because it will break down the plastic incubate 10 mins at room temperature and rinse with DEPC treated H20 Glassware Bake glassware at 300 C for 4 hours or 180 C or higher for several hours Alternatively soak glassware in freshly prepared 0 1 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 4 EZgene Poly Gel RNA Purification Kit Polyacrylamide Gel Recovery Protocol I RNA Probe Elution and Purification 1 Remove the gel from the plastic wrap and place in a 2 0 ug mL acridine orange solution for 15 minutes Destain the gel in distilled water for 10 minutes Re wrap the gel in plastic wrap for easier handling and place the gel on a UV transilluminator to visualize the probe Note Either acridine orange or EtBr may be used for staining 2 Carefully cut the smallest gel fragment which contains the probe with RNase free razor blade Note Cut the gel corresponds to bright purple ba
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