CloneJET™ PCR Cloning Kit
Contents
1. 16 Protocol F Instructions for total RNA purification from bacterial culture up to 109 cells Important Note For RNA isolation bacteria cells should be harvested during the exponential phase of growth ODe 00 0 5 1 Do not use an overnight culture for RNA isolation Before starting e Supplement the required amount of Lysis Buffer with DTT or B mercaptoethanol Add 10 uL 2M DTT or 10 uL 14 3 M B mercaptoethanol to each 450 uL volume of Lysis Buffer required e Supplement the required amount of TE buffer 10 mM Tris HCI pH 8 0 1 mM EDTA with lysozyme not included to final concentration of 0 4 mg mL Note When using the MagJET RNA Kit for the first time e Prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 e Prepare 1X Reaction Buffer with MgCl for DNase as described on page 5 e Transfer the Tissue_RNA_Flex protocol file to the KingFisher Flex or Tissue_RNA_Duo protocol file to the KingFisher Duo 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 RNA Kit can be found on product web page on www thermoscientific com onebio Transfer up to 1 5 mL of bacteria culture up to 1 x 109 cells to a 1 5 mL microcentrifuge tube2. 15 25 C e Wear gloves when handling the Lysis Buffer and Wash Buffer 1 as these reagents contain irritants see p 20 for SAFETY INFORMATION ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED Pipettes and RNase free pipette tips Thermomixer Vortex Microcentrifuge 1 5 mL tubes RNase free Disposable gloves 96 100 ethanol molecular biology grade 2 M DTT or 14 3 M B mercaptoethanol Equipment for sample disruption and homogenization depending on the method chosen e Mortar and pestle Homogenizer Automatic magnetic particle processor and consumables or Magnetic rack Buffers e For mammalian cultured cells lysate preparation PBS 137 mM NaCl 2 7 mM KCI 10 mM Na 2HPO 2 mM KH2POs pH 7 4 e For bacterial lysate preparation TE buffer 10 mM Tris HCI pH 8 0 1 mM EDTA containing lysozyme 0 4 mg mL final concentration e For yeast lysate preparation Yeast lysis buffer 1 M sorbitol 0 1 M EDTA pH 7 4 Add 0 1 8 mercaptoethanol and 5 mg mL of Lyticase or Zymolyase 20T just prior to use AVOIDING RIBONUCLEASE CONTAMINATION RNA purity and integrity is essential for downstream applications RNA can be degraded by RNase A which is a highly stable contaminant found in any laboratory environment Care must be taken not to introduce RNases into the RNA preparation especially during the wash with Wash Buffer 2 and elution steps General recommendations to avoid RNase contamination e Wear gloves when handling
3. Important Note For RNA isolation yeast cells should be harvested at the exponential phase of growth ODe 00 0 5 1 Do not use an overnight culture for RNA isolation For cell disruption using enzymatic lysis described below use only freshly harvested cells Before starting e Prepare Yeast lysis buffer 1 M sorbitol 0 1 M EDTA pH 7 4 Just prior to use add 0 1 B mercaptoethanol and 5 mg mL of Zymolyase 20T Note When using the MagJET RNA Kit for the first time e Prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 e Prepare 1X Reaction Buffer with MgClz for DNase as described on page 5 e Transfer the Tissue_RNA_Flex protocol file to the KingFisher Flex or Tissue_RNA_Duo protocol file to the KingFisher Duo 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 RNA Kit can be found on product web page on www thermoscientific com onebio 1 not provided Collect the cells by centrifugation for 2 min at 212 000 x g Discard the supernatant Transfer the yeast cell culture up to 1 x 108 yeast cells to a 1 5 mL microcentrifuge tube 2 Resuspend the cell pellet in 100 uL of Yeast lysis buffer 3 Incubate at 30
4. ethanol 96 100 per well to the row A to rebind the RNA Row Row name Content Volume per well A DNase Ethanol 200 uL 9 Place the RNA plate back into the instrument and press OK After the pause the protocol will continue to the end 10 After the run is completed remove the RNA plate and Elution Strip according to the instructions on the KingFisher Duo display and turn off the instrument Transfer the eluate which contains the purified RNA to a new sterile tube and close immediately The purified RNA is ready for use in downstream applications Keep purified RNA on ice for immediate use or store at 20 C or 70 C 13 Protocol D Instructions for total RNA purification from up to 20 mg tissues using KingFisher Duo and Microtiter deep well 96 plates Before starting e Supplement the required amount of Lysis Buffer with DTT or B mercaptoethanol Add 10 uL 2M DTT or 10 uL 14 3 M B mercaptoethanol to each 450 uL volume of Lysis Buffer required e Transfer the Tissue_RNA_Duo protocol file to the KingFisher Duo as described on page 7 Ensure you are using the KingFisher Duo 12 pin magnet head and heating block Note When using the MagJET RNA Kit for the first time e Prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 e Prepare 1X Reaction Buffer with MgCl for DNase as described on pag
5. not provided Collect cells by centrifugation for 2 min at 212 000 x g Carefully remove the supernatant leaving the pellet as dry as possible 2 Resuspend the pellet in 100 uL of freshly prepared TE buffer supplemented with lysozyme 0 4 mg mL final concentration Invert the tube several times to mix 3 Incubate the resuspended cells for 5 min at 15 25 C Add 450 uL of Lysis Buffer supplemented with DTT or B mercaptoehtanol Mix thoroughly by vortexing for about 15 s until a homogeneous mixture is obtained Spin down the tube to collect all the drops from the walls of the tube Transfer 450 uL of the prepared lysate to RNA or Sample plate depends on instrument used for further purification For manual RNA purification proceed to Step 2 of the Protocol E Instructions for manual RNA purification from up to 2 x 106 cultured mammalian cells and up to 20 mg tissue on page 15 For automated purification using KingFisher Flex 96 or 5 KingFisher Duo instruments proceed to Step 4 of the Protocol A Instructions for total RNA purification from up to 2 x 106 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 9 or Step 4 of the Protocol C Instructions for total RNA purification from up to 2 x 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 13 17 Protocol G Instructions for total RNA purification from yeast culture up to 108 cells
6. page 7 e Supplement the required amount of Lysis Buffer with DTT or B mercaptoethanol Add 10 uL 2M DTT or 10 uL 14 3 M B mercaptoethanol to each 450 uL volume of Lysis Buffer required Note When using the MagJET RNA Kit for the first time e Prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 e Prepare 1X Reaction Buffer with MgCl2 for DNase as described in the page 5 1 Lysis of Cultured Mammalian Cells a Suspension cells Pellet up to 2x108 cells in an appropriate centrifuge tube for 5 min at 300 x g Discard the supernatant Rinse the cells once with PBS to remove residual growth medium Repeat centrifugation step and discard the supernatant b Adherent cells Remove the growth medium from the cells use up to 2x106 cells Rinse the cells once with PBS to remove residual medium Remove and discard PBS Detach the cells from the culture plate by scraping in appropriate volume of PBS or by trypsinization Transfer the cells into microcentrifuge tube not included and pellet them by centrifugation for 5 min at 300 x g Discard the supernatant 2 Obtain five empty Thermo Scientific Microtiter deep well 96 plates and two empty Thermo Scientific KingFisher 96 KF plates 3 Resuspend collected cells in 450 uL of Lysis Buffer supplemented with DTT or B mercaptoethanol Mix by vortexing to obtain a uniform sus
7. processing type Automation protocols are optimized for KingFisher Flex and KingFisher Duo instruments Note Transfer the Tissue_RNA_Flex protocol file to the KingFisher Flex or Tissue_RNA_Duo protocol file to the KingFisher Duo 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 RNA Kit can be found on product web page on www thermoscientific com onebio Protocol selection guide CEE 5 Leaet_ 2 Sample 5 s2 s2 s MagJET Sample type quantity 3 oe2la2s D purification Page Z 2 2 E 2 E 2 protocol lt lt i 96 e ProtocolA page8 Mammalian cell up to 12 P 12 culture 2x106 cells al deca ai variable e ProtocolE page 15 96 e Protocol B page 11 Tissue ee 12 e Protocol D Page 14 variable ProtocolE Page 15 96 e Protocol F 9 Bacterial up to 10 12 page 17 culture cells variable up to 108 Protocol G Yeast culture p 12 e page 18 cells variable e TOTAL RNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS Protocol A Instructions for total RNA purification from up to 2 x 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates Before starting e Transfer the Tissue_RNA_Flex protocol file to the KingFisher Flex as described on
8. C for 30 min Add 450 uL of Lysis Buffer Mix thoroughly by vortexing or pipetting to obtain a uniform suspension Spin down the tube to collect all the drops from the walls of the tube Transfer 450 uL of the prepared lysate to RNA or Sample plate depends on instrument used for further purification For manual RNA purification proceed to Step 2 of the Protocol E Instructions for manual RNA purification from up to 2 x 106 cultured mammalian cells and up to 20 mg tissue on page 15 For automated purification using KingFisher Flex 96 or RNA purification from up to 2 x 10 cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 9 or Step 4 of the Protocol C Instructions for total RNA Microtiter deep well 96 plates on page 13 18 KingFisher Duo instruments proceed to Step 4 of the Protocol A Instructions for total purification from up to 2 x 10 cultured mammalian cells using KingFisher Duo and TROUBLESHOOTING Problem Possible cause and solution Low RNA yield Too much starting material was used for lysate preparation Reduce the amount of starting material Do not use more tissue or cells than indicated in lysis protocol Starting material was not completely disrupted Reduce the amount of starting material Increase disruption time Incomplete re suspension of magnetic particles Fully resuspend the magnetic particles by vortexing before use Ethanol was not added to the lysate Ma
9. RNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS cccseseeteetneteettes 8 Protocol A Instructions for total RNA purification from up to 2 x 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates cccecscssssesssesesesesesesesesnsescececenenerereseseseseseceeereees 8 Protocol B Instructions for total RNA purification from up to 20 mg of tissue using KingFisher Flex 96 and Microtiter deep well 96 plates ccccccscssscsssssessesesesesesesesssessscsssesescesssseseeceeseeeeseecsuanscananasasasasasaeasereneeesers 11 Protocol C Instructions for total RNA purification from up to 2 x 108 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates scssssssssssesssesssessssseseseeesssesesesessssaserasasereeereessees 12 Protocol D Instructions for total RNA purification from up to 20 mg tissues using KingFisher Duo and Microtter deep well 96 Plates a stra sdei aura aavensedhvcesaancneed eacuemane nen eudentehan taal eaten aden Cantril eanreat 14 Protocol E Instructions for manual RNA purification from up to 2 x 108 cultured mammalian cells and up to ZO MO USSU scala estate E E wed yulet E E il eich auc E 15 Protocol F Instructions for total RNA purification from bacterial culture up to 10 CellS ceeseeeeeteeee 17 Protocol G Instructions for total RNA purification from yeast culture up to 108 Cells eseeeeeeeeeeeeeeeee 18 TROUBLESHOOT IN Gierens scar
10. Thermo SCIENTIFIC PRODUCT INFORMATION Thermo Scientific MagJET RNA Kit K2731 K2732 Read Storage information p 4 upon receipt and store kit components appropriately www thermoscientific com onebio 2731 K2732 Lot 00000000 Expiry Date 00 0000 CERTIFICATE OF ANALYSIS Thermo Scientific MagJET RNA Kit is qualified by isolating RNA from 5 mg of frozen mouse muscle or heart 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 Azg0 ratio of 2 00 3 The functional quality of purified RNA is evaluated by RT PCR Quality authorized by gm Jurgita ilinskien Rev 1 Ill 45 CONTENTS page COMPONENTS OF THEW awe scat cio eM eee Oe A he eet ace UN a ee eas 4 STORAGE aione a Cee eR ot ear a RRR ee ER aR eRe a 4 DESCRIPTION guste ca decdivssacein aren eee veil ecm hone ea hate eine ited a al cede 4 PRINCIPLE 52 02 toaa ba he acon a bass bean aan Gee beats E e eera aata 4 IMPORTANT NOTES kertaa A 5 ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED acs cactisaicuniatatiemeaneuu ae uanade 6 AVOIDING RIBONUCLEASE CONTAMINAT ION s ssssssssssirssssreessstressnttssasttessnttenrnteenarttenrnttnnrrtennrreennrreenrnne 6 STARTING MATERIAL HANDLING AND STORAGE 3 cseacccrtccnssacatenncaramieatanneaandeinwnconnreei x arinaes 7 PROTOCOL SELECTION GUDE aar eaaa eaa 8 Aaa tk on 2 cee ee Aa EAE alt i Soatni ge 7 TOTAL
11. e instrument the protocol will begin 8 When the KingFisher Flex pauses at the dispense step after the DNase digestion step approximately 25 minutes after starting the run remove the DNase I plate from the instrument and add 200 uL of the ethanol 96 100 per well to the DNase plate to rebind the RNA Plate Plate type Plate name Content Volume per well number 2 MlcroMter ESP Wer sip Naeel Ethanol 200 uL 96 plate 9 Place the DNase plate back into the instrument and press Start After the pause the protocol will continue to the end 10 When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and turn off the instrument Transfer the eluate which contains the purified RNA to a new sterile tube and close immediately The purified RNA is ready for use in downstream applications Keep the purified RNA on ice for immediate use or store at 20 C or 70 C 10 Protocol B Instructions for total RNA purification from up to 20 mg of tissue using KingFisher Flex 96 and Microtiter deep well 96 plates Before starting e Transfer the Tissue_RNA_Flex protocol file to the KingFisher Flex as described on page 7 e Supplement the required amount of Lysis Buffer with DTT or B mercaptoethanol Add 10 uL 2M DTT or 10 uL 14 3 M B mercaptoethanol to each 450 uL volume of Lysis Buffer required Note When using the MagJET RNA Kit for the first time e Prepa
12. e 5 1 Lysis of mammalian tissues samples Weigh the tissue use up to 20 mg of fresh or frozen tissue and disrupt the material by one of the following methods a Disruption using a mortar and pestle Place up to 20 mg of tissue use up to 10 mg of spleen tissue into liquid nitrogen and grind thoroughly with mortar and pestle Transfer the tissue powder immediately into a 1 5 mL microcentrifuge tube not included containing 450 uL of Lysis Buffer supplemented with DTT or B mercaptoethanol Mix by vortexing to obtain a uniform suspension Spin down the tubes to collect all the drops from the walls of the tube Note e Transfer the tissue powder to the Lysis Buffer as quickly as possible Leaving the powder without the Lysis Buffer can result in degraded RNA e The homogenized tissue should be directly used for RNA purification and should not be stored e All homogenized material must be thoroughly mixed with the Lysis Buffer and should not be allowed to dry on the walls of the tube this can cause degradation of RNA b Disruption and homogenization using homogenizer Homogenize the tissue according manufacturer recommendations 2 Obtain one empty Thermo Scientific Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo elution strip 3 Transfer the prepared lysate into the row B of the RNA plate Microtiter deep well 96 plate 4 Proceed to Step 4 of the Protocol C Instructions for total RNA purification from
13. e beads have formed a tight pellet Without removing the microcentrifuge tube from the magnetic rack remove and discard the supernatant carefully by using a pipette Make sure that all wash solution is removed 6 Add 700 uL of Wash buffer 2 supplemented with ethanol Mix by vortexing for 1 minute Spin briefly Place the tube in the magnetic rack for 1 minute or until the beads have formed a tight pellet Without removing the microcentrifuge tube from the magnetic rack remove and discard the supernatant carefully by using a pipette Make sure that all wash solution is removed 7 Repeat step 6 8 Add 100 uL of nuclease free water Mix thoroughly by vortexing Spin briefly Incubate tubes in a thermomixer at 60 C 700 900 rpm for 5 minutes Spin briefly Place the tube in the magnetic rack for 1 minute or until the beads have formed a tight pellet Without removing the microcentrifuge tube from the magnetic rack carefully transfer eluate containing RNA to a new sterile microcentrifuge tube Keep on ice for immediate use in downstream applications or store at 20 C 70 C f more concentrated RNA is required the volume of the nuclease free water can be reduced to 50 uL If less concentrated RNA is required the volume of the water can be increased up to 150 uL Incubating at 60 C increases RNA yield If 30 lower yield of RNA is acceptable tubes can be incubated at room temperature in thermomixer 700 900 rpm for 5 minutes
14. e halite ati Ra hd an lg aor ote eae 19 SAFETY INFORMATION crac cscs cue tact ceca ce van vc Becca cee vox Gea ne elie sie ico ee nde 20 COMPONENTS OF THE KIT K2731 K2732 MagJET RNA Kit 96 preps 384 preps Lysis Buffer for MagJET RNA Kit 50 mL 200 mL MagJET Magnetic Beads 3x 1 4mL 2 x 8 5 mL DNase lyophilized 1 vial 1 vial 10X Reaction Buffer with MgClz for DNase 3x 1 mL 9x1mL DNase Reconstitution Buffer 1 mL 2 5 mL Wash Buffer 1 conc for MagJET RNA Kit 40 mL 2 x 80 mL Wash Buffer 2 conc for MagJET RNA Kit 45 mL 3 x 45 mL Water nuclease free 30 mL 125 mL STORAGE DNase lyophilized 10X Reaction Buffer with MgClz for DNase and DNase Reconstitution Buffer should be stored at 20 C upon arrival 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 fast and efficient purification of total RNA from cultured mammalian cells tissues bacteria and yeast The kit utilizes paramagnetic bead technology 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 hi
15. gh quality purified RNA is free of proteins nucleases and other contaminants or inhibitors can be used in a wide range of downstream applications such as RT PCR RT qPCR and other enzymatic reactions See Table 1 for typical total RNA yields from various sources PRINCIPLE The MagJET RNA Kit uses the highly efficient MagJET magnetic particle based technology for nucleic acid purification The whole nucleic acid isolation process combines simple steps of sample lysis RNA binding to the magnetic beads DNA removal washing and elution Purification protocols optimized for automated KingFisher instruments utilize 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 protocol is provided where instead of magnetic particles buffers and other reagents are transferred in each of the protocol steps while magnetic beads remain captured on the wall of the tube using a magnetic rack This allows the kit to be used in various throughput applications using a magnetic rack and manual or automated pipetting equipment Table 1 Typical total RNA yields from various sources Source Quantity RNA yield yg Jurkat 108 cells 10 11 HeLa 108 cells 20 30 COS 7 5 x 105 cells 15 17 M
16. h naa e 96 plate Wash buffer 2 supplemented 700 uL D Wash 2_1 with ethanol E Wash 2 2 Wash buffer 2 supplemented 700 uL with ethanol F Empty Empty Empty G Tip Comb 12 tip comb H Empty Empty Empty For better results avoid storage of DNase in 1X Reaction Buffer with MgClz for DNase at room temperature for extended periods of time It is recommended to fill row A the last It is recommended to prepare the Sample row B after rows C E are filled 5 Fill the KingFisher Duo Elution Strip as follows Elution strip Content Reagent volume per well KingFisher Duo elution strip Water nuclease free 100 uL lf more concentrated RNA is required the volume of the nuclease free water can be reduced to 50 uL If less concentrated RNA is required the volume of the water can be increased up to 150 uL 6 Place a Thermo Scientific KingFisher Duo 12 tip comb into row G of the RNA plate 7 Switch on the KingFisher Duo Start the Tissue_RNA_Duo protocol and load the plate and Elution Strip according to the KingFisher display Ensure that the elution strip is placed in the correct direction into the elution block and that the perforated end is facing towards the user After all plates have been loaded the program will start 8 When the KingFisher Duo pauses at the dispense step after the DNase digestion step approximately 25 minutes after starting the run remove the RNA plate from the instrument and add 200 uL
17. ke sure that ethanol was added to the lysate before loading Sample or RNA plate into the KingFisher Flex or King Fisher Duo instrument Prolonged storage of the sample material Prolonged storage of the sample material may reduce the total RNA yield Too small amount of nuclease free water in elution step There should be an adequate volume of the nuclease free water to cover the magnetic beads completely during the elution step Do not use less nuclease free water than indicated in the protocol Manual protocol only Loss of magnetic beads during manual purification Be careful not remove the magnetic beads during purification using manual protocol Not fully resuspended magnetic beads during binding step Mix in the thermomixer at least 5 minutes or longer if it s necessary to obtain a uniform suspension Not fully dispersed magnetic beads during elution step Make sure the magnetic beads are fully dispersed in nuclease free water during elution step Degraded RNA Inappropriate handling of starting material When purifying RNA from fresh samples place samples in liquid nitrogen immediately after harvesting Proceed to lysis and homogenization as quickly as possible Ensure that samples are frozen in liquid nitrogen immediately after collection and stored at 70 C Thawing of the samples should be avoided until addition of Lysis Buffer RNase contamination To avoid RNase contamination wear gloves during all procedures and change g
18. loves frequently Use sterile disposable RNase free pipette tips Remove RNase contamination from non disposable items and work surfaces Low purity Insufficient washing Insufficient washing causes impurities in the Elution step with nuclease free water Ensure that volumes of the Wash Buffer 1 and 2 are as indicated in the protocol Manual protocol only Not fully removed Wash Buffer 1 or Wash Buffer 2 during Wash steps Make sure that all wash solution is removed during wash steps Magnetic particles in the purified RNA Carryover of the MagJET Magnetic Beads to the Elution step may affect the Azeo Azgo ratio however the magnetic beads in the eluted RNA will not affect downstream applications To remove carryover magnetic particles place eluted sample in the magnetic rack once again Carefully transfer eluate to a clean sterile microcentrifuge tube Magnetic Beads that occasionally remain attached to the tip combs at the end of the process do not affect the total RNA yield as the RNA has already been released into the nuclease free water 19 SAFETY INFORMATION Lysis Buffer for MagJET RNA Kit Xn Harmful Hazard determining components of labeling guanidinium thiocyanate Risk phrases 22 Harmful if swallowed 38 Irritating to skin 41 Risk of serious damage to eyes 52 53 Harmful to aquatic organisms May cause long term adverse effects in the aquatic environment Safety phrases 23 Do not breathe ga
19. nd two empty Thermo Scientific KingFisher Flex 96 KF plates 3 Transfer the prepared lysate to the Sample plate Microtiter deep well 96 plate 4 Proceed to Step 4 of the Protocol A Instructions for total RNA purification from up to 2 x 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 9 for the further purification 11 Protocol C Instructions for total RNA purification from up to 2 x 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates Before starting 1 2 Supplement the required amount of Lysis Buffer with DTT or B mercaptoethanol Add 10 uL 2M DTT or 10 uL 14 3 M B mercaptoethanol to each 450 uL volume of Lysis Buffer required Transfer the Tissue_RNA_Duo protocol file to the KingFisher Duo as described on page 7 Ensure you are using the KingFisher Duo 12 pin magnet head and heating block Note When using the MagJET RNA Kit for the first time 1 Prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 Prepare 1X Reaction Buffer with MgCl for DNase as described on page 5 Lysis of Cultured Mammalian Cells a Suspension cells Pellet up to 2x106 cells in an appropriate centrifuge tube for 5 min at 300 x g Discard the supernatant Rinse the cells once with PBS to remove residual growth medium Repeat centrifuga
20. o remove residual growth medium Repeat centrifugation step and discard the supernatant b Adherent cells Remove the growth medium from the cells use up to 2x10 cells Rinse the cells once with PBS to remove residual medium Remove and discard PBS Detach the cells from the culture plate by scraping in appropriate volume of PBS or by trypsinization Transfer the cells into microcentrifuge tube not included and pellet them by centrifugation for 5 min at 300 x g Discard the supernatant Lysis of mammalian tissues samples Weigh the tissue use up to 20 mg of fresh or frozen tissue and disrupt the material by one of the following methods a Disruption using a mortar and pestle Place up to 20 mg of tissue use up to 10 mg of spleen tissue into liquid nitrogen and grind thoroughly with mortar and pestle Transfer the tissue powder immediately into a 1 5 mL microcentrifuge tube not included containing 450 uL of Lysis Buffer supplemented with DTT or B mercaptoethanol Mix by vortexing to obtain a uniform suspension Spin down the tubes to collect all the drops from the walls of the tube Note e Transfer the tissue powder to the Lysis Buffer as quickly as possible Leaving the powder without the Lysis Buffer can result in degraded RNA e The homogenized tissue should be directly used for RNA purification and should not be stored e All homogenized material must be thoroughly mixed with the Lysis Buffer and should not be allowed to dry
21. on the walls of the tube this can cause degradation of RNA b Disruption and homogenization using homogenizer Homogenize the tissue according manufacturer recommendations 15 2 Add 40 uL of MagJET Magnetic Beads resuspended well by vortexing and 400 uL of ethanol 96 100 to the lysate Mix by vortexing for 5 minutes to obtain a uniform suspension Spin briefly to collect droplets Place the sample in the magnetic rack for 1 minute or until the beads have formed a tight pellet Without removing the microcentrifuge tube from the magnetic rack remove and discard the supernatant carefully by using a pipette Make sure that all of the supernatant is removed 3 Add 200 uL 1X Reaction Buffer with MgCl2 for DNase and 5 pL DNase I Mix gently Spin briefly to collect droplets Incubate the tubes in the thermomixer at 37 C 700 900 rpm for 15 minutes 4 Spin briefly to collect droplets Add 200 uL ethanol 96 100 to the tube Vortex briefly then mix by inverting the tube for 5 minutes Spin briefly Place the sample in the magnetic rack for 1 minute or until the beads have formed a tight pellet Without removing the microcentrifuge tube from the magnetic rack remove and discard the supernatant carefully by using a pipette Make sure that all of the supernatant is removed 5 Add 700 uL of Wash buffer 1 supplemented with ethanol Mix by vortexing for 1 minute Spin briefly Place the sample in the magnetic rack for 1 minute or until th
22. ouse heart 5 mg 4 5 Mouse heart 20 mg 18 20 Mouse liver 5 mg 37 45 Mouse liver 20 mg 140 150 Mouse spleen 5 mg 27 30 Mouse brain 5 mg 4 7 Mouse lung 5 mg 5 9 Mouse kidney 5 mg 12 14 Mouse muscle 5 mg 3 4 E coli 109 cells 23 25 Saccharomyces cerevisiae 108 cells 22 25 IMPORTANT NOTES e Add the indicated volume of ethanol 96 100 to Wash Buffer 1 conc and Wash Buffer 2 conc prior to first use 96 preps 384 preps Wash Buffer 1 Wash Buffer2 Wash Buffer 1 Wash Buffer 2 Concentrated buffer 40 mL 45 mL 80 mL 45 mL Ethanol 96 100 40 mL 180 mL 80 mL 180 mL Total volume 80 mL 225 mL 160 mL 225 mL After preparing each solution mark the bottle to indicate that this step has been completed e To prepare the DNase I solution add 0 55 mL Cat K2731 96 preps kit or 2 2 mL Cat K2732 384 preps kit of DNase Reconstitution Buffer to each vial and incubate at room temperature for 5 minutes Occasional gentle rotation of the vial helps to dissolve the DNase but avoid forceful mixing Do not vortex Store at 20 C e To prepare 1X Reaction Buffer with MgCl for DNase for purification of 1 sample mix 20 uL 10X Reaction Buffer with MgClz for DNase and 180 uL of nuclease free water It is recommended to use freshly prepared buffer e Check all solutions for any salt precipitation before each use Re dissolve any precipitate by warming the solution at 37 C and then equilibrate to room temperature
23. pension Spin down the tubes to collect all the drops from the walls of the tube Transfer the prepared lysate to the Sample plate Microtiter deep well 96 plate 4 Prepare the plates as follows Pale Plate type Plate name Content Volume per well number 1X Reaction Buffer with MgCl for DNase ue 2 DNase DNase 5 uL reconstituted F Wash Buffer 1 3 Microtiter deep well Wash 1 supplemented with 700 uL 96 plate ethanol Wash Buffer 2 4 Wash 2_1 supplemented with 700 uL ethanol Wash Buffer 2 5 Wash 2_2 supplemented with 700 uL ethanol 6 KingFisher Flex 96 Elution Water nuclease free 100 uL 7 Apal Tip Plate For better results avoid storage of DNase in 1X Reaction Buffer with MgCl2 for DNase at roomtemperature for extended periods of time It is recommended to prepare the DNase plate the last 5 Add the following reagents to the Sample plate Plate amber Plate type Plate name Content Volume per well M d Lysed sample 450 uL icrotiter deep we 1 96 plate Sample Magnetic Beads 40 uL Ethanol 400 uL 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 KingFisher Flex 96 KF plate 7 Start the Tissues_RNA_Flex protocol on the KingFisher Flex 96 and load the plates according to the KingFisher display After all the plates have been loaded into th
24. re working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 e Prepare 1X Reaction Buffer with MgCl for DNase as described on page 5 1 Lysis of mammalian tissues samples Weigh the tissue use up to 20 mg of fresh or frozen tissue and disrupt the material by one of the following methods a Disruption using a mortar and pestle Place up to 20 mg of tissue use up to 10 mg of spleen tissue into liquid nitrogen and grind thoroughly with mortar and pestle Transfer the tissue powder immediately into a 1 5 mL microcentrifuge tube not included containing 450 uL of Lysis Buffer supplemented with DTT or B mercaptoethanol Mix by vortexing to obtain a uniform suspension Spin down the tube to collect all the drops from the walls of the tube Note e Transfer the tissue powder to the Lysis Buffer as quickly as possible Leaving the powder without the Lysis Buffer can result in degraded RNA e The homogenized tissue should be directly used for RNA purification and should not be stored e All homogenized material must be thoroughly mixed with the Lysis Buffer and should not be allowed to dry on the walls of the tube this can cause degradation of RNA b Disruption and homogenization using homogenizer Homogenize the tissue according manufacturer recommendations 2 Obtain five empty Thermo Scientific Microtiter deep well 96 plates a
25. reagents and RNA samples as skin is a common source of RNases Change gloves frequently e Use sterile disposable RNase free pipette tips e Use appropriate reagents to remove RNase contamination from non disposable items such as pipettes centrifuges and work surfaces e Keep all kit components tightly sealed when not in use After use close bottles immediately STARTING MATERIAL HANDLING AND STORAGE e When purifying RNA from fresh samples place the samples in liquid nitrogen immediately after harvesting Proceed to lysis and homogenization as quickly as possible e When samples are obtained from sacrificed animals or cadavers limit the time between death and sample collection to isolate high quality RNA e If RNA is not to be purified immediately after tissue collection freeze the samples in liquid nitrogen and store at 70 C Frozen tissue samples should not be allowed to thaw during handling or weighing e Mammalian cultured cells can be pelleted and stored at 70 C until required e For RNA purification from yeast cells using enzymatic lysis only freshly harvested samples can be used PROTOCOL SELECTION GUIDE The MagJET RNA Kit provides optimized protocols for total RNA purification from mammalian cultured cells tissue bacteria and yeast The kit is compatible with automated and manual processing The following selection guide summarizes available protocols depending on the source of starting material throughput and sample
26. s fumes vapour spray 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 39 Wear suitable protective clothing gloves and eye face protection 60 This material and its container must be disposed of as hazardous waste 61 Avoid release to the environment Refer to special instructions safety data sheets Wash Buffer 1 for MagJET RNA Kit Xn Harmful Hazard determining components of labeling guanidinium chloride Risk phrases 22 Harmful if swallowed 36 38 Irritating to eyes and skin Safety phrases 3 Keep in a cool place 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 All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries 20
27. tion step and discard the supernatant b Adherent cells Remove the growth medium from the cells use up to 2x10 cells Rinse the cells once with PBS to remove residual medium Remove and discard PBS Detach the cells from the culture plate by scraping in appropriate volume of PBS or by trypsinization Transfer the cells into microcentrifuge tube not included and pellet them by centrifugation for 5 min at 300 x g Discard the supernatant Resuspend collected cells in 450 uL Lysis Buffer supplemented with DTT or B mercaptoethanol Mix by vortexing to obtain a uniform suspension Spin down the tubes to collect all the drops from the walls of the tube Obtain one empty Thermo Scientific Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo elution strip Transfer the prepared lysate to row B of the RNA plate 12 4 Prepare the RNA plate Microtiter deep well 96 plate according to the instructions below Add the following reagents to the rows Note that row G is reserved for the tip and should be left empty Note that rows F and H are left empty Plate name and Row Rowinamne Content Volume per type well 1X Reaction Buffer with MgClo 200 uL A DNase for DNase DNase reconstituted 5 uL Lysed Sample 450 uL a Magnetic Beads resuspended 40 uL B Sample before use Ethanol 400 uL RNA plate Wash buffer 1 supplemented 700 uL Microtiter deep well C Wash 1 wit
28. up to 2 x 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 13 for the further purification 14 Protocol E Instructions for manual RNA purification from up to 2 x 106 cultured mammalian cells and up to 20 mg tissue Following protocol is based on transfer of liquids by pipetting through different purification steps rather than magnetic bead transfer as in KingFisher automatic protocols This allows the kit to be used in various throughput applications using magnetic rack and manual or automated pipetting equipment Protocols for the other automated pipetting platforms should be optimized for each platform and sample used To enable protocol optimization all buffers are available to purchase separately Before starting e Supplement the required amount of Lysis Buffer with DTT or B mercaptoethanol Add 10 uL 2M DTT or 10 uL 14 3 M B mercaptoethanol to each 450 uL volume of Lysis Buffer required Note When using the MagJET RNA Kit for the first time e Prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Reconstitute DNase lyophilized in DNase Reconstitution Buffer as described on page 5 Prepare 1X Reaction Buffer with MgClz for DNase as described on page 5 1 Lysis of Cultured Mammalian Cells a Suspension cells Pellet up to 2x10 cells in an appropriate centrifuge tube for 5 min at 300 x g Discard the supernatant Rinse the cells once with PBS t
Download Pdf Manuals
Related Search