MagJET Plant RNA Kit - Thermo Fisher Scientific
Contents
1. ccccssseeeeeessesseeeeeees 10 Protocol B Instructions for total RNA purification from up to 50 mg plant sample using KingFisher Duo with 12 pin magnet head and Microtiter deep well 96 plate 12 Protocol C Instructions for manual plant total RNA purification from up to 50 mg Eo AAA E O O a name 14 TROUBLE SOO TIN o eee dnd 15 SAFETY INFORMATION ciar 16 COMPONENTS OF THE KIT MagJET Plant RNA Kit cid oa Lysis Buffer for MagJET Plant RNA Kit 72 mL 2 x 140 mL MagJET Magnetic Beads 2 1 4mL 10 6 mL DNase lyophilized 1 vial 4 vials DNase Reconstitution Buffer 1 mL 2x1mL 2X DNase Buffer 12 mL 45 mL Manganese Chloride Solution 3x 1mL 9x1mL Wash Buffer 1 conc for MagJET Plant RNA Kit 110 mL 3 x 110 mL Wash Buffer 2 conc for MagJET Plant RNA Kit 50 mL 3 x 50mL Rebinding Buffer for MagJET Plant RNA Kit 20 mL 70 mL Water nuclease free 30 mL 125 mL STORAGE When the kit is delivered remove DNase lyophilized DNase Reconstitution Buffer and Manganese Chloride Solution and store at 20 C Reconstituted DNase should be stored at 20 C MagJET Magnetic Beads should be stored at 4 C Other components of the kit should be stored at room temperature 15 25 C DESCRIPTION The MagJET RNA Kit is designed for automated high throughput or manual purification of total RNA from a wide variety of plant species and tissue types The kit utilizes paramagnetic bead techno2. Rowname Content Volume per well Plant RNA plate Rebinding Buffer conc 150 uL Microtiter deep well 96 A DNase Ethanol plate 96 100 AOp 9 Place the Plant RNA plate back into the instrument and press OK After the pause the protocol will continue through to completion 10 After the run is complete remove the Plant RNA plate and Elution strip according to the instructions on the KingFisher Duo display and turn off the instrument Transfer the purified RNA from the Elution strip to a fresh RNase free microtiter plate or microcentrifuge tubes Keep on ice for immediate use in downstream applications or store at 70 C Protocol C Instructions for manual plant total RNA purification from up to 50 mg plant sample Following this protocol is based on the transfer of liquids by pipetting through different purification steps rather than magnetic bead transfer as in the KingFisher automatic protocols This allows the kit to be used in various throughput applications using a magnetic rack and manual or automated pipetting equipment Protocols for automated pipetting platforms should be optimized for each platform and sample type To enable protocol optimization all buffers are available for purchase separately Note When using the MagJET Plant RNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 and reconstitute DNase as described on page 5 1 Homogenize the plant samples and prepare
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4. 100 ae 96 plate D Wash 1_1 Wash Buffer 1 700 uL E Wash 1_2 Wash Buffer 1 700 uL F Wash 2_3 Wash Buffer 2 700 uL G Wash 2_4 Wash Buffer 2 700 uL H Empty Empty Empty Note For best results avoid storing DNase master mix at room temperature for long term lt is recommended to prepare the DNase row A after filling Wash buffers rows D E F G It is recommended to prepare the Sample row C last 5 Fill the KingFisher Duo Elution strip as follows Elution strip Content Volume per well KingFisher Duo Elution strip Water nuclease free 100 uL 6 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on the Plant RNA plate 7 Switch on the KingFisher Duo instrument and ensure you are using the KingFisher Duo 12 pin magnet head and heating block Start the Plant_RNA_Duo protocol Insert the Plant RNA plate and Elution strip into the instrument as indicated on the KingFisher Duo display and press START Make sure that the Elution strip is placed in the correct direction into the elution block ensure that the perforated end is facing toward the user 8 When the KingFisher Duo pauses at the dispense step after the DNase digest approximately 30 minutes after starting the run remove the Plant RNA plate from the instrument and add 150 uL of Rebinding Buffer concentrated and 400 uL ethanol 96 100 per well to row A DNase row to rebind the RNA Plate name and type Row
5. PRODUCT INFORMATION Thermo Scientific MagJET Plant RNA Kit K2771 K2772 Read Storage information p 4 upon receipt and store kit components appropriately www thermoscientific com onebio K2771 Lot 00000000 Exp 00 0000 CERTIFICATE OF ANALYSIS Thermo Scientific MagJET Plant RNA Kit is qualified by isolation of total RNA from 50 mg of plant tissue following the protocol outlined in the manual The quality of isolated RNA is evaluated spectrophotometrically and by agarose gel electrophoresis The purified RNA has an Azeo Azgo ratio of 2 0 0 2 and the RNA integrity number RIN of 27 Quality authorized by Jurgita Zilinskien Rev 1 Ill 69 CONTENTS page COMPONEN TSG TRE osa id 4 O AA eee AT E E RU CES EIDE OMIT rer EAC iE E A RII Eater E OE 4 DESCRIPTION uva io es 4 PRINCIPLES oscila ieee eae 4 IMPORTANT NOTES ostia dll does 5 ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED o ococcccccccccococococooooooooooooooooononooo20 6 AVOIDING RIBONUCLEASE CONTAMINATION cccecssesesssssssssesescscsssssseessnsnssssesssesesersees 6 RNA HANDLING AND STORAGE consi 6 STARTING MATERIAL HANDLING AND SAMPLE HOMOGENIZATION c ccccssssssssssseseeees 7 PROTOCOL SELECTION GUIDE dl a dd create 9 TOTAL RNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS 10 Protocol A Instructions for total RNA purification from up to 50 mg plant sample using KingFisher Flex 96 and Microtiter deep well 96 plates
6. by RNase A which is a highly stable contaminant found in any laboratory environment Care must be taken not to introduce RNases into RNA preparations especially during wash with Wash Buffer 2 and elution steps General recommendations to avoid RNase contamination include the following Wear gloves when handling reagents and RNA samples as skin is a common source of RNases Change gloves frequently e Use sterile disposable RNase free pipette tips Use appropriate reagents to remove RNase contamination from nondisposable items pipettes centrifuges and work surfaces Keep all kit components tightly sealed when not in use After usage close bottles immediately RNA HANDLING AND STORAGE Keep the RNA on ice after extraction and while working with it Store the extracted RNA at 20 C or 70 C For long term use store RNA at 70 C STARTING MATERIAL HANDLING AND SAMPLE HOMOGENIZATION To minimize RNA degradation avoid repeated freezing and thawing of the samples and perform extractions from fresh material or material that has been immediately frozen and stored at 70 C Appropriate sample storage is essential for reproducibility and high RNA yields Yields of RNA may vary depending on sample age type of sample and storage conditions Efficient homogenization of the sample material is an essential step before RNA purification in order to gain a good yield of high quality RNA The lysis procedure of plant tissue is most effective
7. by vortexing and briefly spin to remove droplets from the inside of the lid Place the tube on the magnetic stand and let the magnetic beads collect at the magnet for 2 3 minutes Discard the supernatant using a pipette 8 Wash 2 remove the tube from the magnetic stand and add 700 uL Wash Buffer 1 supplemented with ethanol see page 5 Resuspend the magnetic beads by vortexing place the tube on the magnetic stand and let the magnetic beads collect at the magnet for 2 3 minutes Discard the supernatant using a pipette 9 Wash 3 remove the tube from the magnetic stand and add 700 uL Wash Buffer 2 supplemented with ethanol see page 5 Resuspend the magnetic beads by vortexing place the tube on the magnetic stand and let the magnetic beads collect at the magnet for 2 3 minutes Discard the supernatant using a pipette 10 Wash 4 repeat step 9 using 700 uL of Wash Buffer 2 Make sure that all the supernatant is completely removed in the last washing step 11 Dry leave the tube on magnetic rack for 5 min to dry the magnetic beads at room temperature Remove the tube from the magnetic rack 12 Elution add 100 uL Nuclease free water Resuspend the magnetic beads by vortexing briefly spin then place the tube on the magnetic stand and let the magnetic beads collect at the magnet for 2 3 minutes 13 While on the magnetic stand transfer the eluate which contains the purified RNA to a new RNase free tube then close immediately Keep on ice
8. centrifuges and work surfaces RNA degraded during sample storage Make sure the plant samples were properly stored and make sure the samples were processed immediately after collection or removal from storage RNA degraded during sample homogenization To minimize RNA degradation avoid repeated freezing and thawing of the samples and perform extractions from fresh material or material that has been immediately frozen and stored at 70 C Transfer the ground tissue to the Lysis Buffer for MagJET Plant RNA Kit as quickly as possible to avoid RNA degradation All ground material must be thoroughly mixed with the Lysis Buffer for MagJET Plant RNA RNA degradation can occur in particles that are left to dry on the walls of the tube Lysis Buffer was not supplemented with DTT Ensure that 2 M DTT solution has been added to the Lysis Buffer before RNA purification procedure Purified RNA was not stored properly Purified RNA should be used immediately in downstream applications or stored at 20 C for later use For prolonged storage more than 1 month storage at 70 C is recommended Inhibition of downstream enzymatic reactions Purified RNA contains residual salt Use the correct order of Wash Buffers steps Always wash the magnetic beads with Wash Buffer 1 first and then proceed with Wash Buffer 2 Purified RNA contains residual ethanol It is important to dry magnetic beads before elution step Genomic DNA contamination Ineffici
9. eded e Check all solutions in the kit for salt precipitation before each use Re dissolve any precipitates by warming the solution at 37 C and then equilibrate to room temperature 20 5 C e Wear gloves when handling the Lysis Buffer Rebinding Buffer and Wash Buffer 1 as these reagents contain irritants see page 16 for SAFETY INFORMATION ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED 2 M DTT dithiotreitol solution store at 20 C polyvinylpyrrolidone PVP for RNA purification from woody lignified and or polyphenol rich samples see p 7 8 Sodium chloride NaCl for RNA purification from soy beans see p 7 8 96 100 ethanol molecular biology grade Pipettes and RNase free pipette tips 8 or 12 channel pipettes and pipette tips RNase free 1 5 2 mL plastic tubes Disposable gloves Thermomixer Centrifuge capable of gt 16 000 x g for microtubes Centrifuge capable of 3 000 4 000 x g with swinging bucket rotor for 96 well plates Incubator or water bath capable of 56 C Vortex Mortar and pestle for manual tissue disruption in liquid nitrogen Liquid nitrogen Commercial homogenizer and stainless steel beads for mechanical automatic plant tissue disruption Automatic magnetic particle processor and consumables For manual protocol acquire magnetic particle processing rack AVOIDING RIBONUCLEASE CONTAMINATION RNA purity and integrity is essential for downstream applications RNA can be degraded
10. ent DNase digestion Ensure that the temperature of DNase digestion is 37 C Prepare DNase mix according to protocol see p 7 and p 10 Too much sample input if starting amount of plant sample is too high gt 50 mg the DNA digestion step may not be effective Carryover of the magnetic beads in the elution Carryover of magnetic beads in the eluted RNA will not affect downstream applications To remove the carryover from eluted RNA simply magnetize the magnetic beads and carefully transfer eluate to a new tube or plate 15 SAFETY INFORMATION Lysis Buffer for MagJET Plant RNA Kit Xn Harmful Hazard determining components of labelling guanidinium thiocyanate Risk phrases R20 21 22 Harmful by inhalation in contact with skin and if swallowed R32 52 53 Contact with acids liberates very toxic gas Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Precautionary statements P261 Avoid breathing dust fume gas mist vapours spray P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P301 P312 IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell P304 P340 IF INHALED Remove victim to fresh air and keep at rest in a position comfortable for breathing P501 Dispose of contents con
11. entific KingFisher Flex 96 KF plates 4 Fill the plates as follows Plate Volume per Number Plate type Plate name Content well a Wash Buffer 1 2 Microtiter deep well 96 plate Wash 1_1 supplemented with ethanol 700 uL 3 Microtiter deep well 96 plate DNase DNase master mix 200 uL ae Wash Buffer 1 4 Microtiter deep well 96 plate Wash 1_2 supplemented with ethanol 700 pL as Wash Buffer 2 5 Microtiter deep well 96 plate Wash 2_3 supplemented with ethanol 700 uL ae Wash Buffer 2 6 Microtiter deep well 96 plate Wash 2_4 supplemented with ethanol 700 uL 7 KingFisher Flex 96 KF plate Elution Water nuclease free 100 uL 8 KingFisher Flex 96 KF plate Tip plate For best results avoid storage of DNase in 1X Reaction Buffer with MgCl2 at room temperature for extended periods of time It is recommended to prepare the DNase plate just prior to use 5 Prepare the Sample plate Microtiter deep well 96 plate Add the following reagents to the Sample plate Piae Plate type Plate name Content volume per number well Plant tissue lysate 400 uL 1 Microtiter deep well 96 plate Sample Magnetic Beads aoe Ethanol 400 uL 96 100 M Resuspend Magnetic Beads well by vortexing before use 6 Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a Tip Plate an empty KingFisher Flex 96 KF plate 7 Start the Plant_RNA_Flex protocol
12. for immediate use in downstream applications or store at 70 C 14 TROUBLESHOOTING Problem Possible cause and solution Low yield of purified RNA RNA degraded during sample storage Make sure the plant samples were properly stored and make sure the samples were processed immediately after collection or removal from storage Insufficient homogenization of plant material To disrupt the cell wall it is important to homogenize the sample thoroughly until it is ground to a fine powder Ethanol was not added to the lysate Ensure that ethanol was added to the lysate during bindind to magnetic beads step Incomplete resuspension of magnetic particles Resuspend the magnetic particles by vortexing before use Ethanol was not added to Wash Buffer 1 add ethanol to Wash buffer 1 as described on page 5 Ethanol was not added to Wash Buffer 2 ensure that ethanol was added to Wash Buffer 2 as described on page 5 Rebinding step was missed ensure that rebinding buffer and ethanol was added after DNase treatment Loss of magnetic beads during purification use sufficient magnetic beads capture time be careful not remove magnetic beads during procedures Purified RNA is degraded RNase contamination To avoid RNase contamination wear gloves during the procedure and change gloves frequently Use sterile disposable RNase free pipette tips Use reagents designed to remove RNase contamination from nondisposable items pipettes
13. is Buffer with sodium chloride NaCl at a 2 M final concentration Mechanical automatic tissue disruption Plant tissue can be homogenized with commercial homogenizers and grinding mills with stainless steel beads High throughput homogenizers offer a suitable method for handling up to 96 samples simultaneously 1 ow Place up to 50 mg of plant sample or up to 20 mg for seeds into microtube containing a Stainless steel bead and 600 uL Lysis Buffer supplemented with DTT as described on page 5 Grind the sample according to homogenizer instructions Incubate for 5 min at 56 C Centrifuge for 10 min at maximum speed 216 000 x g for microtube or 3 000 4 000 x g for racked 96 well collection microtubes Carefully take 400 uL of the cleared supernatant and immediately proceed with the RNA isolation according to protocols A Notes for lysis of different plant samples Use 10 50 mg of fresh plant sample for mechanical automatic disruption with stainless steel beads in single microtubes Use 10 20 mg plant Isample for mechanical automatic disruption with stainless steel beads in racked 96 well collection microtubes Use 10 20 mg of plant seeds as starting material for mechanical automatic disruption with Stainless steel beads in racked 96 well collection microtubes and or single mictrotubes To purify total RNA from woody lignified and or polyphenol rich samples such as branches twigs needles wax c
14. logy enabling high yields and robust performance High binding capacity uniform particle size and rapid magnetic response of MagJET magnetic beads makes the technology ideal for high throughput automatic nucleic acid purification as well as for manual nucleic acid purification by low sample throughput users The resulting high quality purified RNA is free of proteins nucleases and other contaminants or inhibitors and can be used in a wide range of downstream applications such as RT PCR RT gPCR and other enzymatic reactions See Table 1 for typical total RNA yields from various sources PRINCIPLE The MagJET Plant RNA Kit uses the highly efficient MagJET magnetic particle based technology for nucleic acid purification The nucleic acid isolation process combines the simple steps of sample lysis RNA binding to the magnetic beads DNA digestion washing and elution Purification protocols optimized for automated KingFisher instruments utilize a high throughput magnetic bead transfer technique where magnetic beads are transferred through different reagent plates containing lysis binding washing and elution reagents This enables high throughput nucleic acid purification and eliminates multiple pipetting steps Alternatively a protocol is provided where buffers and other reagents are transferred in each of the protocol steps while magnetic beads remain captured on the wall of a tube using a magnetic rack This allows the kit to be used in vari
15. lysates as described on page 7 8 SAMPLE HOMOGENIZATION 2 Carefully transfer 400 uL of lysate supernatant to a clean microcentrifuge tube 3 Binding to magnetic beads add 25 uL MagJET Magnetic beads resuspended well by vortexing and 400 uL 96 100 ethanol Vortex the tube and briefly spin to remove droplets from the inside of the lid Place the tube on the magnetic stand and let the magnetic beads collect at the magnet for 2 3 minutes or until the solution clears Discard the supernatant using a pipette 4 Wash 1 remove the tube from the magnetic stand and add 700 uL Wash Buffer 1 supplemented with ethanol see page 5 Resuspend the magnetic beads by vortexing briefly spin then place the tube on the magnetic stand and let the magnetic beads collect at the magnet for 2 3 minutes Discard the supernatant using a pipette 5 Dry leave the tube on the magnetic rack for 5 minutes to dry the magnetic beads at room temperature Remove the tube from the magnetic rack 6 DNase treatment add 200 uL of DNase solution for each purification mix 100 uL 2X DNase Buffer 4 uL DNase reconstituted 20 uL Manganese Chloride Solution and 76 pL nuclease free water Prepare common mix for all samples to be processed Mix and incubate in the thermomixer at 37 C and 350 rpm for 15 minutes 7 Rebinding to magnetic beads after DNase treatment add 150 uL of Rebinding Buffer concentrated and 400 uL 96 100 ethanol Resuspend the magnetic beads
16. oated leaves e g laurel supplement the Lysis Buffer with polyvinylpyrrolidone PVP at a 2 w v final concentration To purify total RNA from soy beans supplement the Lysis Buffer with sodium chloride NaCl at a 2 M final concentration Proceed according homogenizer manufacturer recommendations The MagJET Plant RNA Kit provides optimized protocols for total RNA purification from PROTOCOL SELECTION GUIDE different amounts of starting material 10 50 mg The Kit is compatible with automated and manual sample processing allowing high and low throughput nucleic acid purification workflows The following selection guide summarizes the available protocols depending on starting sample weight throughput and sample processing method Automation protocols are optimized for KingFisher Flex and KingFisher Duo instruments Protocol selection guide S 4 O Plant Be 55 58 35 MagJET ci ample mae LES 2 amp 33 P homogenization 5 S5553 purification Page t weight 203 sli 5 28 ype E o 3 E E E 2 protoco lt lt samedi d Up to 96 o ProtocolA 10 with liquid 10 50 mg nitrogen see 10 20 mg Up to 12 o ProtocolB 12 the detailed of seeds instruction on variable e ProtocolC 14 page 7 Plant honiogenized Up to 96 o ProtocolA 10 with equipment l 10 50 m for disrupting 10 20 id Up to 12 e ProtocolB 12 plant tissue of seeds see the detailed instruction on variable o Pro
17. ous throughput applications using a magnetic rack and manual or automated pipetting equipment Table 1 Typical yields of total RNA from various sources Plant Tissue 50 mg RNA yield ug Arabidopsis thaliana leaf 18 20 Nicotiana tabacum leaf 15 20 Tomato leaf 21 52 Barley seedlings leaf 25 32 Wheat shoots leaf 25 36 Spinach leaf 24 30 Onion leaf 8 12 Rice leaf 16 19 Corn leaf 13 20 seeds 20 40 leaf 11 19 Canola Rape stalk 22 28 seeds 30 Soy leaf 30 40 seeds 44 Cucumber fruit 18 Potato stalk 10 Sunflower stalk seeds 40 IMPORTANT NOTES e Add the indicated volumes of ethanol 96 100 to Wash Buffer 1 conc and Wash Buffer 2 conc prior to first use K2771 and K2772 Wash Buffer 1 Wash Buffer 2 Concentrated buffer 110 mL 50 mL Ethanol 96 100 110 mL 200 mL Total volume 220 mL 250 mL After preparing each solution mark the bottle to indicate that this step has been completed e To prepare the DNase solution add 0 44 mL of DNase Reconstitution Buffer to each vial of DNase lyophilized and incubate at room temperature for 5 minutes Occasional gentle rotation of the vial helps to dissolve the DNase I but avoid forceful mixing Do not vortex Store at 20 C e Before each RNA purification prepare a fresh aliquot of Lysis Buffer supplemented with 2 MDTT solution Add 20 uL of 2 M DTT to each 1 mL of Lysis Buffer ne
18. tainer in accordance with local regional national international regulations Wash Buffer 1 conc for MagJET Plant RNA Kit Xn Harmful Hazard determining components of labelling guanidinium chloride Risk phrases 22 Harmful if swallowed 36 38 Irritating to eyes and skin Safety phrases 23 Do not breathe gas fumes vapour spray 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 Wear suitable protective clothing and gloves 60 This material and its container must be disposed of as hazardous waste x Rebinding Buffer for MagJET Plant RNA Kit Xn Harmful Hazard determining components of labelling guanidinium chloride Risk phrases 22 Harmful if swallowed 36 38 Irritating to eyes and skin Safety phrases 23 Do not breathe gas fumes vapour spray 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 Wear suitable protective clothing and gloves 60 This material and its container must be disposed of as hazardous waste PRODUCT USE LIMITATION This product is developed designed and sold exclusively for research purposes and in vitro use only The product was not tested for use in diagnostics or for drug development nor is it suitable for administration to humans or animals Please refer to www thermoscientific com onebio for Material Safety Data Sheet of the product 2013 Thermo Fisher Scientific Inc All rights reserved
19. tocol C 14 page 8 Sample quantity may depend on the homogenizer type and throughput Please proceed according homogenizer manufacturer recommendations TOTAL RNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS Protocol A Instructions for total RNA purification from up to 50 mg plant sample using KingFisher Flex 96 and Microtiter deep well 96 plates Note 3 When using the MagJET Plant RNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 and reconstitute DNase as described on page 5 Transfer the Plant_RNA_Flex protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 2 User Manual The protocol files for MagJET Plant RNA Kit can be found on the product web page on www thermoscientific com onebio Homogenize the plant samples and prepare lysates as described on page 7 8 SAMPLE HOMOGENIZATION Prepare DNase master mix for the number of samples to be processed DNase master mix for purification of one sample 200 uL 100 uL 2X DNase Buffer 4 uL DNase reconstituted 20 uL Manganese Chloride Solution and 76 uL nuclease free water Aliquot prepared master mix to the DNase I plate plate number 3 below Obtain six empty Thermo Scientific Microtiter deep well 96 plates and two empty Thermo Sci
20. u are using the KingFisher Duo 12 pin magnet head and heating block e Transfer the Plant_RNA_Duo protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 2 User Manual The protocol files for MagJET Plant RNA Kit can be found on product web page on www thermoscientific com onebio 1 Homogenize the plant samples and prepare lysates as described on page 7 8 SAMPLE HOMOGENIZATION 2 Prepare DNase master mix for the number of samples to be processed DNase master mix for purification of one sample 200 uL 100 uL 2X DNase Buffer 4 uL DNase reconstituted 20 uL Manganese Chloride Solution and 76 uL nuclease free water Aliquot prepared master mix to the Microtiter deep well 96 plate row A DNase row 3 Obtain one empty Thermo Scientific Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo Elution strip 4 Prepare the Plant RNA plate Microtiter deep well 96 plate Add the following reagents to the rows Note that row B is reserved for the tips and should be left empty Note that row H is left empty Plate name and type Row Row name Content Sample reagent volume per well A DNase DNase master mix 200 uL B Tip 12 tip comb N A Plant tissue lysate 400 uL c Sample Magnet Beads 25 uL ano Microtter doop wel 96
21. with the KingFisher Flex 96 and load the plates according to the KingFisher display After all the plates have been loaded into the instrument the protocol will begin 8 When the KingFisher Flex pauses at the dispense step after the DNase digestion approximately 30 minutes after starting the run remove the DNase plate from the instrument and add 150 uL of Rebinding Buffer concentrated and 400 uL of ethanol 96 100 per well to rebind the RNA Plate Volume per Amber Plate type Plate name Content well Rebinding Buffer conc 150 uL 3 Microtiter d Il 96 plate DNase icrotiter deep well 96 plate ase Ethanol 400 uL 96 100 9 Place the DNase plate back into the instrument and press Start After the pause the protocol will continue through to completion 10 After the protocol is complete remove the plates according to the instructions on the KingFisher Flex display and turn off the instrument Transfer the purified RNA from the Elution plate to a fresh RNase free microtiter plate Keep on ice for immediate use in downstream applications or store at 70 C Protocol B Instructions for total RNA purification from up to 50 mg plant sample using KingFisher Duo with 12 pin magnet head and Microtiter deep well 96 plate Note e When using the MagJET Plant RNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 and reconstitute DNase as described on page 5 e Ensure yo
22. with well homogenized powdered samples Plant tissue can be homogenized using a mortar and pestle in the presence of liquid nitrogen or using commercial homogenizers with steel beads Manual plant tissue homogenization 1 2 OO amp 00 Place the plant sample in a mortar with liquid nitrogen Grind the tissue using a pestle Allow the liquid nitrogen to evaporate and transfer the powder sample 10 50 mg into a 1 5 mL microcentrifuge tube containing 600 uL Lysis Buffer supplemented with DTT as described on page 5 e Transfer the homogenized tissue to the Lysis Buffer as quickly as possible to avoid RNA degradation e All homogenized material must be thoroughly mixed with the Lysis Buffer RNA degradation can occur in particles that are left to dry on the walls of the tube Vortex for 10 20 s to mix thoroughly Incubate for 5 min at 56 C Centrifuge for 10 min at maximum speed 216 000 x g Carefully take 400 uL of the cleared supernatant and immediately proceed with the RNA isolation according to protocols A C Notes for lysis of different plant samples For seed samples use 10 20 mg starting material into the Lysis Buffer To purify total RNA from woody lignified and or polyphenol rich samples such as branches twigs needles wax coated leaves such as laurel supplement the Lysis Buffer with polyvinylpyrrolidone PVP at a 2 w v final concentration To purify total RNA from soy beans supplement the Lys
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