Total RNA Purification from Plant

Transcription

1 H A N D B O O K TM RNASure Plant Kit NP Preps RNASure Plant Kit NP Preps Genetix Bitech Asia Pvt. Ltd. 71/1, First Flr, Shivaji Marg, Najafgarh Rad, New Delhi Phne : Fax : inf@genetixbitech.cm TM

2 TablefCntents COMPONENTS Kit cntents Reagents, cnsumables, and equipment nt supplied with the kit SAFETYINSTRUCTIONS INTRODUCTION Principle and Prcedure Specificatins f RNASure Plant Kit Starting Material and Typical yield Hmgenizatin f Plant samples Preparatin f reagents fi PROTOCOLSFORNASUREPLANTKIT SUPLEMENTARYPROTOCOLFORrDNaseDIGESTIONINSOLUTION TROUBLESHOTING ORDERINGINFORMATION PRODUCTWARANTY ***** [ 1 ]

3 KIT CONTENTS Reagents, cnsumables, and equipment nt supplied with the kit RNA Sure Plant Cat. N. NP NP Number f Preps Wash Buffer RWB2 15 ml 80 ml Lysis Buffer RLB1 25 ml 125 ml Lysisi Buffer RLB-P 25 ml 125 ml Wash Buffer RWB3 (cnc)* 12.5 ml 3 x 25 ml Desalting Buffer RDB 25 ml 125 ml Reactin buffer fr rdnase 7 ml 35 ml rdnase,rnase-free (lyphilised)* 1 vial (size D) 1 vial (sized) RNase-free Water 15 ml 65 ml RNASure Shredder RNASure Plant Clumn Cllectin tubes(2ml) Cllectin tube (1.5ml) Handbk 1 1 * Please see Preparatin f Reagents % ethanl (t prepare Wash Buffer RWB3) 70 % ethanl (t adjust RNA binding cnditins) ß-mercaptethanl, r DTT (dithithreithl), 1.5 ml micrcentrifuge tubes Sterile RNase-free pipette tips Manual pipette Centrifuge fr micrcentrifuge tubes Equipment fr sample disruptin and hmgenizatin (see sectin 2.3) Persnal prtectin equipment (lab cat, glves, gggles) SAFETY INSTRUCTIONS When wrking with chemicals, always wear a suitable lab cat, dispsable glves, and prtective gggles. Fr mre infrmatin, please cnsult the apprpriate material safety data sheets (MSDSs). Buffers RLB1,RLB-P and RWB2 cntain guanidine salts, which can frm highly reactive cmpunds when cmbined with bleach. If liquid cntaining these buffers is spilt, clean with suitable labratry detergent and water. If the spilt liquid cntains ptentially infectius agents, clean the affected area first with labratry detergent and water, and then with 1% (v/v) sdium hypchlrite. The fllwing risk and safety phrases apply t cmpnents f the RNASure Plant Kit: Buffer RLB1 Cntains Guanidium thicyanate: R&S Phrases: R20/21/22, S13 Buffer RLB-P Cntains Guanidium hydrchlride: R&S Phrases: R22-36/38 Buffer RWB2 Cntains Guanidium thicyanate: R&S Phrases: R10-20/21/22, S Desalting Buffer RDB Cntains Guanidium thicyanate <10% + ethanl <10%: R&S Phrases: R10, S7-16 rdnase Cntains lyphilized rdnase: R&S Phrases: R42/43, S22-24 R10: Flammable, R20/21/22: Harmful by inhalatin, in cntact with skin and if swallwed, R36/38: Irritating t eyes and skin, May cause sensitizatin by inhalatin and skin cntact, R42/43: May cause sensitizatin by inhalatin and skin cntact, S7: Keep cntainer tightly clsed, S13: Keep away frm fd, drink and animal fdstuffs, S16: Keep away frm surces f ignitin - N smking, S22: D nt breathe dust, S24: Avid cntact with skin. [ 2 ] [ 3]

4 INTRODUCTION Principle and Prcedure RNASure Plant Mini Kit prvide a fast and easy way t purify DNA frm plant and fungal tissue. Up t 100 mg f tissue can be prcessed using the DNeasy Plant Mini Kit. Cells are disrupted by grinding in presence f liquid nitrgen. The lysed samples are then incubated with buffers cntaining chatrpic salts which are capable f prtecting RNA frm endgenus RNAses. Cntaminatin f DNA is remved by n-clumn RNase free rdnase treatment. The samples are then prcessed thrugh a spin cartridge cntaining clear silica based membrane t which RNA binds. Any impurities are remved by subsequent tw washing steps. Pure RNA is then eluted with RNase-free water supplied with the kit, which can further be used in variety f dwnstream applicatins. The RNASure Plant kit cntain tw different lysis buffers RLB1 and RLB-P respectively. Fr all general applicatins buffer RLB1 is recmmended fr Lysis. In certain plant tissue and fungi peculiar metablites are present, then buffer RLB is ptimized fr lysis f samples cntaining peculiar metablites. All RNA preparatin steps can be perfrmed at rm temperature. The final eluate shuld be kept frzen at -20 C fr shrt term strage and keep it at -70 C fr lng term strage t prevent RNA degradatin. Specificatins f RNASure Plant Kit RNASure Plant kit is simple reliable and rapid methd fr the islatin f ttal RNA frm plant cells and tissues r filamentus fungi. Within 30 minute high quality f RNA can be prepared frm 10mg f plant tissues. If used in RT-PCR applicatins we recmmend using intrn-spannig primers. The kit is supplied with rdnase fr an n-clumn digestin t have minimal DNA cntaminatin. An ptinal digestin with rdnase can be perfrmed fr mst demanding applicatins. Starting material and typical yield Upt 70ìg f RNA can be extracted frm 100mg f starting material with an elutin vlume f 60ìl using RNASure Plant Mini Kit. Hmgenizatin f Plant Samples Plant Tissues Cmplete disruptin f cell walls and plasma membranes f cells and rganelles is abslutely required t release all the RNA cntained in the sample. Depending upn the sample type disruptin can be dne mechanically. Fr disruptin using a mrtar and pestle, freeze the animal tissue immediately in liquid nitrgen and grind t a fine pwder under liquid nitrgen. Transfer the suspensin (tissue pwder and liquid nitrgen) int a liquid-nitrgen cled, apprpriately sized tube and allw the liquid nitrgen t evaprate withut allwing the sample t thaw. Add lysis buffer RLB1 cntaining beta mercaptethanl and mix immediately. Then pass lysate thrugh RNASure Shredder (included in the kit ) r thrugh 0.9mm syringe needle. At least 5 10 times r until a hmgeneus lysate is achieved. Increasing the vlume f lysis buffer may be required t facilitate handling and minimize lss. Rtr statr hmgenizers disrupt and hmgenize simultaneusly in the presence f lysis buffer RLB1, single samples f animal tissues in secnds depending n the tughness and size f the sample. Rtr statr hmgenizers can als be used t hmgenize cell lysates. The rtr turns at a very high speed, causing the sample t be disrupted and hmgenized by a cmbinatin f turbulence and mechanical shearing. Faming f the sample shuld be kept t a minimum by using prperly sized vessels, keeping the tip f the hmgenizer submerged, and hlding the immersed tip t the side f the tube. Rtr statr hmgenizers are available in different sizes and perate with differently sized prbes. Prbes with diameters f 5 mm and 7 mm are suitable fr vlumes up t 300 ìl and can be used fr hmgenizatin in micrcentrifuge tubes. Prbes with a diameter f 10 mm r abve require larger tubes. In additin, rund-bttmed tubes allw mre efficient hmgenizatin than cnical bttmed. Maintain RNase free envirnment during the entire prcess. Preparatin f Reagents Always wear glves & safety gggles as buffers cntain guanidine salts. All buffers & kit cmpnents are stable at rm temperature (18 C-25 C) fr at least 12 mnths, Lyphilized rdnase (RNase free) at 4 C n arrival (it is stable upt 12 mnths). Keep up 70% ethanl ready fr use in prtcl. Recnstitute Wash Buffer RWB3 Recnstitute Buffer RWB3 cncentrate by adding, 50ml (Kit NP-84905) and 100ml t each bttle (Kit NP-84907) ethanl (96-100%). Mark the bttle as Ethanl Added. Buffer RWB3 can be stred at rm temperature (18-25 C) fr at least ne year. rdnase Add 540ul f RNase Freewater t each rdnase vial and incubate fr ne minute at rm temperature (18-25 C). gently mix t disslve the rdnase cmpletely. Dispense int aliquts and stre at -20 C. the frzen sample is stable fr 6 mnths. D nt freeze thaw mre than three times. [ 4 ] [ 5]

5 RNA Purificatin frm Plant Hmgenized Plant Sample Lysis 100 mg tissue 350 µl Buffer RLB1 3.5 µl â-mercaptethanl Mix Prtcl fr Ttal RNA islatin frm plant tissue r Filamentus fungi Ttal RNA purificatin with RNASure Plant Kit Befre starting the preparatin: Check if Wash Buffer RWB3 and rdnase were prepared as per instructins Lysate Filtratin Prcedure 1. Add liquid nitrgen t RNase free mrtar and grind well upt 100mg tissues. Hmgenize upt 100mg f plant material in a precled mrtar and pestle and grind well. Binding Desalting Digest DNA with DNase Wash 1 Wash 2 Wash 3 Elutin Add 350 ìl Ethanl (70%) t the filtrate, Mix and Lad 11,000 g, 1 min 11,000g, 1 min 350 µl Buffer RDB1 11,000g, 1 min 95 µl DNase RT, 15 min 200 µl RWB2 buffer 11,000g, 30 sec 600 µl RWB3 buffer 11,000g, 30 sec 200 µl RWB3 buffer 11,000g, 2 min 60 ìl RNase-free H 2 O 2. Add 350µl buffer RLB1 and 3.5µl ß-mercaptethanl t the hmgenized 100mg f tissues frm step 1 and vrtex well. Nte: As alternative t ß-ME the reducing agent DTT r TCEPmay be used. Use a final cncentratin f mm DTT r TCEP within the Lysis Buffer RLB1 r RLB. 3. Place the RNASure Shredder in cllectin tube (2ml), apply the mixture and centrifuge at 11,000 x g fr 1 min. Transfer the filtrate t a new centrifuge tube 1.5 ml capacity ( nt prvided but available frm genetix brand ). Nte : D nt disturb the pellet f cell debris, which may be visible after centrifugatin. Transfer the pellet t a new 1.5ml centrifuge tube (nt prvided but available frm genetix brand). Discard the shredder clumn and prceed with step 4 4. T the abve slutin add 350µl f ethanl (70%) and mix thrughly by pulse vrtexing (10-15s). Nte: Sme time after additin f ethanl a stringy precipitate may becme visible which will nt affect the RNA islatin. Be sure t disaggregate any precipitate by mixing and lad all f the precipitate n the clumn as described in step 5. D nt centrifuge the ethanlic lysate befre lading it nt the clumn in rder t avid pelleting the precipitate. 5. Take a new RNASure Plant Clumn and place in cllectin tube and lad the lysate. Centrifuge at 11,000 x g fr 30 secnds. Then place the clumn in new cllectin tube (2ml). Maximum lading capacity f RNA Sure Plant Clumns are 750 µl. Repeat the prcedure if larger vlumes are t be prcessed 6. Add 350 µl RDB and centrifuge at 11,000 x g fr 1 min t dry the membrane. Salt remval will help the fllwing rdnase digest much mre effective. If the clumn utlet has cme int cntact with the flw-thrugh fr any reasn, discard the flw-thrugh and centrifuge again fr 30 s at 11,000 x g. 11,000g, 1 min Purified Ttal RNA [ 6 ] [ 7]

6 7. Fr n clumn DNase digestin prepare DNase reactin mixture (by adding 10ul f DNase t 90µl f DNase reactin buffer in a 1.5ml micrcentrifuge tube). Mix prperly and apply 95µl f it directly nt the centre f silica membrane f the RNASure Clumn (prvided). Incubate atr rm temperature fr 15 minutes. 8. T the RNASure clumn add 200µl f buffer RWB2 and centrifuge fr 30 secnds at x g discard the filtrate. This step will inactivate the rdnase. Place the clumn in new cllectin tube. 9. T the RNA Sure clumn add 600µl f buffer RWB3 & Centrifuge fr 30 Secnds at 11,000 x g. Nw discard the filtrate. Transfer the clumn int a new cllectin tube. 10. Add 200µl f buffer RWB3 t the clumn & centrifuge it fr 2-3 min at x g this step will dry the membrane. Place the clumn in new nuclease free 1.5 ml cllectin tube (supplied). If fr any reasn, the liquid level in the cllectin tube has reached the RNASure Plant clumn after centrifugatin, discard flw-thrugh, and centrifuge again. 11. Elute the RNA in 60 µl RNase-free H2O, (supplied) and centrifuge at 11,000 x g fr 1 min. If higher RNA cncentratins are desired, elutin can be dne with 40 µl but the yield will decrease slightly. Supplementary Prtcl fr rdnase digestin in slutin rdnase digestin in slutin Althugh,n clumn rdnase digestin is very efficient in DNA remval. But in certain applicatin even traces f DNA need t be remved, prceed with rdnase treatment in slutin. Prepare rdnase as per instructins Prepare rdnase by adding 10µl f RNase Free rdnase t 90µl f rdnase Buffer. Add 6µl f it t 60µl f eluted RNA (1/10th vlume f extracted RNA). Incubate the mixture fr 10 min at 37 C. Repurify the RNA with a suitable RNA cleanup prcedure (see manufacturer's prtcl f respective kit) r ethanl precipitatin. Ethanl Precipitatin T ne vlume f sample add 1/10th vlume f 3M sdium Acetate ph 5.2 and 2.5 vlumes f % chilled ethanl. Incubate 30minutes at - 20 C r 4 C. Centrifuge fr 10 min at 11000xg. Wash RNA pellet with 70 % ethanl. Dry RNA pellet and resuspend RNA in RNase-free H O. 2 TROUBLESHOOTING GUIDE RNA is Degraded/ N RNA is btained RNase cntaminatin Use sterile individually wrapped plastic wares Use nly sterile, dispsable RNase free pipette tips and micrcentrifuge tube. Wear dispsable glves and keep changing the glves frequently Always use prper micrbilgical technique Maintain RNase free wrking envirnment Glassware shuld be ven baked fr at least 2 hurs at 25 C befre use Pr RNA Quality r yield Fllw prtcl guidelines f each sample type, buffers preparatin etc Mix and vrtex reagent prperly Binding f RNA is effective in presence f ethanl nly make sure t add ethanl t lysis buffer Imprper strage f kit cmpnents T much starting Material T much f starting material may give lw yield. If require buffer RLB1 can be increased. Clear hmgenate and remve any particulate by use f RNASure Shredder Clumns. gdna Cntaminatin rdnase slutin nt prperly applied Pipette rdnase slutin directly t the center f the silica membrane withut wetting the rim rdnase nt active Recnstitute and stre lyphilized rdnase as per instructin Perfrm ptinal DNase digestin step during the sample preparatin r after purificatin. Use intrn-spanning primer if pssible. [ 8 ] [ 9 ]

7 Subptimal perfrmance f RNA in dwnstream experiments Stre islated RNA prperly Finally eluted RNA prperly shuld always be kept at - 20 C and fr lng term strage freeze at -70 C. Presence f ethanl r salt Increase centrifugatin time during wash steps Check flw-thrugh des nt tuch the clumn utlet after the secnd buffer RWB3 wash. Use crrect rder f wash buffer. ORDERINGINFORMATION Descriptin PackSize Cat.N. DNASure Tissue Mini Kit 50 preps NP DNASure Plant Mini Kit 50 preps NP DNASure Plant Mini Kit 250 preps NP DNASure Plant Midi Kit 20 preps NP DNASure Plant Maxi Kit 10 preps NP DNASure Bld Mini Kit 50 preps NP DNASure Bld Mini Kit 250 preps NP DNASure Bld Midi Kit 20 preps NP DNASure Bld Maxi Kit 10 preps NP DNASure Bld FastPure Kit 50 preps NP DNASure Bld FastPure Kit 250 preps NP SureSpin Plasmid Mini Kit 50 preps NP SureSpin Plasmid Mini Kit 250 preps NP SureSpin Plasmid FastPrep Kit 50 preps NP SureSpin Plasmid FastPrep Kit 250 preps NP SureSpin Buffer Set* BS SurePrep Plasmid Mini Kit 20 preps NP SurePrep Plasmid Mini Kit 100 preps NP SurePrep Plasmid Midi Kit 20 preps NP SurePrep Plasmid Midi Kit 100 preps NP SurePrep Plasmid Maxi Kit 10 preps NP SurePrep Plasmid Maxi Kit 25 preps NP SurePrep Plasmid Mega Kit 5 preps NP SurePrep Plasmid Giga Kit 5 preps NP fi *SureSpinBuferSet Fr the islatin f lw-cpy plasmids, buffers PA1, PA2, PA3, RNase A, sufficient fr 300 preps [ 10 ] [ 11 ]

8 ORDERINGINFORMATION Descriptin PackSize Cat.N. SurePrep Buffer Set** BS SurePrep Plasmid Endfree Maxi Kit 10 preps NP SurePrep Plasmid Endfree Mega Kit 5 preps NP SurePrep Plasmid Endfree Giga Kit 5 preps NP SureSpin 96 PCR Kit 4x96 NP SureTrap Gel Extractin Kit 50 preps NP SureTrap Gel Extractin Kit 250 preps NP SureTrap PCR Cleanup Kit 50 preps NP SureTrap PCR Cleanup Kit 250 preps NP SureExtract Spin PCR/Gel Extractin Kit 50 preps NP SureExtract Spin PCR/Gel Extractin Kit 250 preps NP SureSEQ Cleanup Kit 50 preps NP RNASure Mini Kit 50 preps NP RNASure Mini Kit 250 preps NP RNASure Plant Kit 50 preps NP RNASure Plant Kit 250 preps NP mirnasure Mini Kit 50 preps NP SureTrap mrna Mini Kit 12 preps NP SureTrap mrna Midi Kit 12 preps NP RNASure Virus Kit 50 preps NP RNASure Virus Kit 250 preps NP PrductWaranty RNASure Plant Kit cmpnents are intended fr research purpses nly. They are suitable fr in vitr uses nly. The purchaser must determine the suitability f the prduct fr its particular use. Shuld any prduct fail t perfrm satisfactrily due t any reasn ther than misuse, Genetix will replace it free f charge r refund the purchase price. Genetix reserve the right t change, alter, r mdify any prduct t enhance its perfrmance and design. It is the respnsibility f the user t verify the use f the RNASure Plant Kit fr a specific applicatin range as the perfrmance characteristic f this kit has nt been verified t a specific rganism. N claim r representatin is intended fr its use t identify any specific rganism r fr clinical r therapeutic use. Genetix des nt warrant against damages r defects arising in shipping and handling (transprt insurance fr custmers excluded), r ut f accident r imprper r abnrmal use f this prduct. In accrdance with Genetix ISO-certified Quality Management System, each lt f RNASure Plant Kit is tested against predetermined specificatins t ensure cnsistent prduct quality. In n event shall Genetix be liable fr claims fr any ther damages, whether direct, indirect, incidental, cmpensatry, freseeable, cnsequential, r special (including but nt limited t lss f use, revenue r prfit), whether based upn warranty, cntract, trt (including negligence) r strict liability arising in cnnectin with the sale r the failure f Genetix prducts t perfrm in accrdance with the stated specificatins. Prduct claims are subject t change. Therefre please cntact ur Technical Supprt Department fr updated infrmatin n Genetix prducts. Please cntact: Genetix Bitech Asia (P) Ltd. 71/1, Najafgarh Rad, Shivaji Marg, New Delhi INDIA. inf@genetixbitech.cm techsupprt@genetixbitech.cm Tel: Fax: Trademarks: RNASure is a registered trademark f Genetix Bitech Asia (P) Ltd. fi *SureSpinBuferSet Fr islatin f lw-cpy plasmids, csmids, BACs, PACs, and P1 cnstructs, nly applicable with SurePrep Plasmid kits, sufficient fr 10 SurePrep Maxi Clumns (Maxi preps), 20 SurePrep Midi Clumns (Midi preps), set incl. RNase A [ 12 ] [ 13 ]

9 NOTE: NOTE: [ 14 ] [ 15 ]

10 NOTE: [ 16 ]