User`s Manual - Gene-Foci
Contents
1. bene foc Gene Foci Biotechnologies EASYspin RNA Extraction Kit Catalog No GF2704 User s Manual For Research Use Only In vitro Use Only se rec Oe EASYspin RNA Extraction Kit Catalog No GF2704 Catalog No Preps GF2704 50 50 GF2704 200 200 APPLICATIONS Ideal for fast RNA extraction from cells and tissue samples Kit Contents And Storage Conditions Storage PCR Purification Kit a 50 preps 200 preps Conditions Lysis Buffer RLT Room Temp 50 ml 200 ml Buffer RW1 Room Temp 40 ml 160 ml 10 mi 40 ml Wasi Buter RNY noom Temp Add ethanol before first use RNase free H20 Room Temp 10 ml 40 ml 9 ml RNase free H2O 36 ml RNase free H gt O 70 Ethanol Room Temp Add ethanol before first use RNase free Columns RA Room Temp 50 200 and Collection Tubes This kit can be stored at room temperature for up to 12 months without showing any decrease in quality and yield gt AS NOTES All buffers should be clear Lower temperature may cause precipitation If any precipitation forms warm at 37C water bath to dissolve before use The Easyspin RNA Extraction kit should be stored at room temperature store at 4 C or 20 C may cause chemical compound precipitation in buffers Recap the bottles immediately after use to avoid unexpected oxidation evaporation and change of pH due to long term exposure to the air INTRODUCTION The Easyspin RNA Extraction Kit offers a2. 50 ul 600 pl adjust the ethanol volume accordingly if some lysate is lost during the above procedure and pipet to mix immediately Precipitation may be formed after the addition of ethanol but this does not affect the procedure Do not centrifuge Transfer up to 700 pl mixture into a RNA binding column RA column placed in a 2 ml collection tube provided Centrifuge at 13 000 rpm for 30 s and discard the flow through Repeat this step if the sample volume exceeds 700 ul Add 700 ul Buffer RW1 and incubate at room temperature for 30s Centrifuge at 12 000 rpm for 30s Discard the flow through Incubate for 5 min at room temperature before centrifugation if obvious DNA remains Add 500 gul Buffer RW and centrifuge at 12 000 rpm for 30 s Discard the flow through Repeat Step 6 with another 500 pl Buffer RW Place the RA column back into the same collection tube Centrifuge the empty RA column at 13 000 rpm for 2 min to completely remove ethanol from the column Place the RA column in a RNase free microcentrifuge tube Add 30 50 ul of RNase free water pre warm the water to 70 90 C will increase the RNA yield to the center of the column membrane Incubate at room temperature for 1 min and centrifuge at 12 000 rpm for 1 min to elute the RNA If the expected RNA yield is gt 30 yg repeat step 8 with another 30 50 wl of RNase free water or using the eluate from step 8 if high RNA concentration is reguired Reuse the centrifuge tub
3. amount of water immediately To prevent RNase contamination the following precautions should be taken when handling RNA 1 Change gloves frequently to avoid RNase contamination from the skin 2 Use RNase free plasticware and tips to avoid cross contamination 3 RNA will not be degraded in Buffer RLT Plus But in the subsequent steps RNase free plasticware and glassware should be used Glassware should be oven baked at 150 C for 4 hr Plasticware can be treated with 0 5 M NaOH for 10 min followed by thorough rinse with water and autoclave 4 Use RNase free DEPC treated water to prepare solutions add DEPC to water at a final concentration of 0 1 v v and leave at 37 C overnight and autoclave RNA detection Integrity of RNA The integrity of the purified RNA can be detected by agarose gel electrophoresis 1 2 agarose gel 0 5x TBE buffer The ribosomal RNA rRNA should appear as sharp bands on the ethidium bromide stained gel ble gt Oe tae _ se rec 2 Oo under UV 28S rRNA bands should be present with the intensity approximately twice that of the 18S rRNA band If the rRNA bands appear as a smear of smaller sized RNAs it is likely that the RNA sample is degraded during preparation Purity of RNA The ratio of OD gt eo0 OD2so provides an estimate of the purity of RNA with respect protein contamination However the OD2e0 ODo2so ratio is influenced considerably by pH Lower pH res
4. e from step 8 The RNA yield should be 15 30 higher if using a second volume of RNase free water than that obtained using the eluate from step 8 however the final RNA concentration will be lower Ordering Information To order Gene Foci products please try the following methods 1 Order online Register for an account on www Gene Foci com login and place your order using our shopping cart and secure online checking out system 2 Call our toll free number 1 888 315 9018 3 Send Email to order Gene Foci com 4 Fax your order to 1 888 959 0868 To expedite your order please provide the following information Customer user name Purchaser s name and detailed contact information Purchase Order Number If any Billing address Shipping address Description of the order
5. ell pellet thoroughly by flicking the tube Add 350 pl lt 5x10 cells or 600 pl 5x10 1x10 cells of Buffer RLT Plus pipet or vortex to mix d Homogenization cells lt 1x10 can be homogenized by vortexing for 1 min Pass the lysate at least 5 times through a 20 gauge needle 0 9 mm diameter fitted to an RNase free syringe Homogenization shears genomic DNA reduces the viscosity of the lysates and increases the yields e Immediately proceed to Step 3 2 Animal soft tissues for example mouse liver and brain a Mince fresh tissues into small pieces add 350 pl lt 20 mg tissue or 600 il 20 30mg tissue of Buffer RLT Plus Homogenize with electronic tissue homogenizer for 20 40 s Or b Immediately place the tissue in liquid nitrogen and grind thoroughly with a mortar and pestle Transfer adequate amount 20mg 30mg of tissue powder in to a 1 5 ml microcentrifuge tube containing 350 yl 600 pl of Buffer RLT vortex for 20 s Pass the lysate at least 5 times through a 20 gauge needle 0 9 mm diameter fitted to an RNase free syringe or homogenize with an electronic tissue homogenizer This step shears genomic DNA reduces the viscosity of the lysates and increases the yields De SK eo Ss Oo Soe rec oO c Centrifuge the homogenized lysate at 13 000 rpm for 3 min Transfer the supernatant into a new centrifuge tube d Immediately proceed to Step 3 Add equal volume of 70 ethanol usually 3
6. fast and easy way to extract high quality RNA from tissues and cells The whole precess is phenol chloroform free The unique lysis buffer immediately lyses biological samples and inactivates RNase and DNase Ethanol is added to the lysate to provide appropriate binding conditions for RNA and RNA selectively binds to the silica membrane of the RNA column in the high salt buffer RNA is purified through a series of wash spin steps to remove protein followed by elution of RNA from silica membrane with RNase free H20 gt 1 HIGHLIGHTS High quality silica membranes are used to ensure the yield and consistency between different batches Fast and convenient The whole RNA purification process from one sample can be done within 30 min Multiple washing steps guarantee high quality RNA purification The OD260 280 of G se SKe ce gt Oo Eye _ rs rec 2 Oo gt the RNA product is typically between 1 8 2 1 ATTENTION All the steps should be performed at room temperature use microcentrifuge such as Eppendorf 5415C or similar model that can handle 13 000 rpm or higher speed Materials and reagents to be supplied by the user ethanol 2 mercaptoethanol single use syringes mortar and pestle Lysis buffer RLT and wash buffer RW1 contain irritating chemicals wear gloves when handling Avoid direct contact with skin eyes and clothes If contaminated rinse with large
7. ults in a lower OD260 OD280 ratio and reduced sensitivity to protein contamination In 10mM Tris pH7 5 pure RNA has a OD2e0 OD2g0 ratio of 1 8 2 1 In water the ratio is 1 5 1 9 and this does not mean the RNA is not pure Ouantification of RNA Dilute the RNA sample with RNase free water and measure the OD260 using a spectrophotometer which has been calibrated with RNase free water The concentration of RNA sample ng ul is calculated using the following formula OD gt eo x 40 x dilution factor G le oO EASYSPIN RNA EXTRACTION KIT PROTOCOL Please read Attention part before start Hints Before the first use add the indicated amount of ethanol into buffer RW and 70 ethanol bottles mix well and mark the bottle with a check Before the first use please add 2 mercaptoethanol to Buffer RLT to achieve a final concentration of 1 working solution For example add 10 ul of 2 mercaptoethanol per 1ml Buffer RLT This mixture can be stored at 4C for one month Procedure 1 Culture cells a Harvest lt 10 cells grown in suspension into a centrifuge tube Adherent cells can be lysed directly in cell culture vessels or trypsinized from culture flasks and collected into a centrifuge tube b Centrifuge at 13 000xg for 10 sec or 300xg for 5min to pellet cells Completely aspirate the supernatant Note Incomplete removal of the supernatant will decrease the yield and purity c Loosen the c
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