Instructions For Use
Contents
1. BCSI SNAP Simple Nucleic Acid Processing Instructions for Use For Research Use Only Blood Cell Storage Inc 454 North 34 Street Seattle WA 98103 478 227 4005 JULY 12 Rev 01 Contents Product Informal 3 BCESISNAP Producida ld idea 3 E O E ETES 3 Equipment and Reagents Supplied by the User eee ceeesseeeescecsaeceeeeseeesaeecaecsaeesseeseneessaeens 3 Product Use Limitations siniese rnai a E A ETA EE 4 Quality Control AR 4 Technical Assistante a A Na 4 Satety Information es ca senssenes cha ianceaseearahostanacaeateaactaswacaveqsvaseaatsaacgeedunees EE a IA 4 General Procedure for Nucleic Acid Isolati0N ooonnncccnoncccnnncccnoncnonnncnononcncnnnnnonnncnononcncnnncnnonnnccnnnnnos 5 Reagent Preparation ni aE E EE TE E A AE RE 5 Ca TAINS E E 6 Manta Washing Of GANS 35 alt a O O 7 Manual Elton ls sad 7 Automated and WAS A ne 9 Automated A O 9 MOTT ENS GALSTAD aos 9 Applicaton Noli dd 11 BESI Product Maui untada aiii iaa 11 Total Nucleic Acids from Pla Ms O 11 Total Nucleic Acids from FFPE Tissue Sample is 13 Total Nucleic Acid from E COll ini na E a 14 Vital RNA Brom Plasma dan 15 Appendix Risk andes ate ly Phrases SO NS 16 Product Information BCSI SNAP Products Core Components SNAP Ca R f O Oaaa 24 SNAP Lysis GT B ffer onn a ee i adici n A 30 mL SNAP Lysis GT ND Buffer 15 mL SNAP Wash 1 Concentrate _s 60 mL SNAP Wash 2 Co2. bubbles Repeat as needed to fill the card 2 Wipe any residual sample or sample deposits from the surface of the card Direction of wiping should be away from the exit port Take care to avoid contaminating the exit port 3 Incubate at room temperature to allow binding of nucleic acids Note The binding time will rely on the sample type being analyzed Refer to Application Notes for suggested binding times p ooo O Exit Port Manual Washing of Cards 1 With a wide bore pipet tip remove the sample from the card through the exit port Try to remove as much residual sample as possible A CAUTION Do not attempt to decontaminate biological samples with bleach Samples containing guanidine may be autoclaved 2 Fill the channel through the entry port with prepared Wash 1 Let sit for one 1 minute and then remove through the exit port Repeat for a total of two washes Remove as much residual Wash 1 as possible 3 Fill the channel with prepared Wash 2 Let sit for one 1 minute and then remove through the exit port Repeat for a total of 6 washes Remove as much residual Wash 2 as possible 4 Place cards in vacuum desiccator until the interior surface is visually dry 5 Proceed to manual elution To facilitate manual card processing the SNAP M kit is available from BCSI The SNAP M kit enables gravity driven flow to simplify the wash process and includes a quick and easy drying
3. 0 5 mL of Resuspension buffer Vortex to obtain an even suspension From a liquid culture Transfer a sample of the liquid culture to a 1 7 mL tube Pellet the bacteria by spinning in a microcentrifuge for 2 minutes Suspend the bacterial pellet in 0 5 mL Resuspension buffer using a vortex mixer to obtain an even suspension Add 1 drop SNAP Protease 2 solution and mix well Allow the sample to digest for 30 minutes at room temperature Extraction Procedure Add 1 mL SNAP GT Lysis buffer to the sample The E coli suspension will become clear on GT buffer addition Load the sample onto an extraction card as outlined in detail above Let the card sit at room temperature for 30 minutes Wash and dry the card as directed above Elute bound nucleic acid with 100 200 ul Elution Buffer 14 Viral RNA From Plasma 1 Required Reagents 1 BCSI SNAP GT ND Lysis 2 BCSI SNAP Protease 1 3 BCSI SNAP Carrier 1 2 Procedure 1 Pipet 0 5ml of sample containing phage into a 1 7ml microcentrifuge tube NOTE If less than 0 5ml of sample is to be used add pure water or additional UVTM to a final volume of 0 5ml and follow the procedure below Add 1 drop 40ul protease Mix well either by vortexing the mixture and spinning briefly to collect clinging drops in the cap or by using a pipetor Incubate at room temperature for at least 30 minutes NOTE If the sample is water the protease step is optional Add 0 5ml of GTND Buffer and 0 5m
4. AP Carrier 1 is used in the extraction protocol to increase the yield of very small quantities of target RNA However the use of carrier is optional It may be helpful to test the effectiveness of carrier in the user defined application Each tube of carrier contains 1 mg of dry poly rA RNA Add 200 ul of elution buffer Mix well Let sit for a few minutes and mix well once again It is best to prepare smaller aliquots of the carrier RNA and store these at 20 C for up to one year A CAUTION DO NOT add bleach or acidic solutions to the sample preparation waste The Lysis Buffer and Wash 1 solutions contain high concentrations of Guanidine which can form toxic compounds when combined with bleach Card Loading This section describes how to load a prepared sample into a BCSI Extraction Card To determine the sample preparation appropriate for your application please refer to the Application Notes section of this document The procedure described below is for a 1 5 mL total volume containing 0 5 mL sample 0 5 mL lysis buffer and 0 5 mL ethanol The channel in the isolation card has a total volume of 1 2 1 5 mL However lower volumes can be loaded onto the card 1 With a 1 mL pipetor fixed with a wide orifice pipet tip load on the entire sample through the entry port Ensure there is a tight seal with the pipette tip and the entry port to prevent sample leakage Slowly depress plunger to load the sample Try not to introduce excessive
5. SDS Combine all in the 1 7 mL tube 4 Add 50 ul SNAP Protease 2 Incubate overnight at 55 C follow by a 70 C heat pulse for one hour 5 Add 600 ul SNAP GT Lysis Buffer and mix well 6 Proceed to Section 5 below 4 Nucleic Acid Isolation on BCSI Extraction Cards 1 Load the entire sample onto an extraction card There is less volume of sample at this point Sample Volume 0 9 mL than can fit in the channel Total Volume 1 5 mL Move the sample over the entire surface of the glass to completely coat the channel and park the sample over the center of the card Let the nucleic acids adsorb to the glass for at least 30 minutes Longer adsorption times may increase the yield 3 Wash and dry the cards as directed 4 Elute the bound nucleic acids using 100 ul elution buffer 13 Total Nucleic Acid from E coli Note This procedure is for extraction of total nucleic acid from isolated colonies or broth cultures of E coli Extraction of other bacterial species or of E coli from other sample types may require further optimization Extraction of gram positive species and yeasts will require additional enzymatic digestion to increase the efficiency of extraction 1 Required Reagents 1 2 3 BCSI SNAP GT Lysis BCSI SNAP Protease 2 Dissolved to 5 mg mL Resuspension Buffer contains 1 SDS Sodium dodecyl sulfate 2 Sample Preparation From an agar plate Scrape up an appropriate number of colonies into
6. ard Elution The machine will introduce the elution buffer and move it most of the way through the channel When the machine protocol finishes the eluate will be located within the S channel With a 1 mL pipette tip carefully move the eluate to the end of the channel nearest the entry port by pushing from the exit port Once it has reached the end of the glass portion of the channel reverse its flow and pull it back through the channel to remove the eluate through the exit port to a clean tube On The Card Storage BCSI SNAP Cards offer stable storage of purified Nucleic Acids at room temperature To take advantage of this feature simply stop processing after the card drying step Note if using the SNAP R automated wash station ensure it is not programmed to automatically introduce the elution Once dry the cards can be stored at room temperature To recover your NA simply elute as usual at a later date RNA can be recovered up to seven days after washing and DNA can be recovered up to six months after washing BCSI Nucleic Acid Extraction System Guide Removal of Eluate After Automated Elution Step 1 Remove card from wash station Elution bubble is located away from o i the Entry Port EZ o Step 2 With a pipettor at the Exit Port a push the elution bubble towards the end of the channel nearest the Entry Port gt Step 3 With a pipettor at the Exit Port o draw the elution bubble bac
7. cal Assistance www bloodcellstorage com support Ph 478 BCSI 005 478 227 4005 M F 09 00 17 00 PST Email na bloodcellstorage com Safety Information When working with chemicals always wear appropriate Personal Protective Equipment PPE If any of the reagents are spilled clean the affected area with the use of absorbent towels or wipes Rinse the affected areas with water as appropriate Biological samples may be decontaminated by autoclaving Disposal of unused reagents and biological samples should be according to local regulations Refer to Appendix A for reagent specific safety information For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www bloodcellstorage com on the Technical Support page where you can find view and print the MSDS for each BCSI kit and kit components Further information is also provided on the last page of this booklet General Procedure for Nucleic Acid Isolation Reagent Preparation NOTE To ensure reagent purity DO NOT mix residual reagents from previous kits with fresh reagents being prepared SNAP Lysis Buffer SNAP Lysis Buffer contains a high concentration of guanidine salts in solution If the reagent has experienced low temperatures some guanidine may come out of solution Before use check to see that the reagent is without precipitate If precipitate is present it may be necessary to wa
8. d elution buffer sample from the previous elution onto the second card Move it to the opposite port and allow to sit for 10 20 minutes 6 Slowly bring the elution buffer carefully back across the channel and remove The sample is now ready to use 12 Total Nucleic Acids from FFPE Tissue Samples 1 Required Reagents BCSI SNAP GT Lysis BCSI SNAP Protease 2 PinPoint Solution Zymo Research Catalog Number D3001 1 Xylene Pure Ethanol 100 0 1 SDS in water Dilute a 10 SDS Solution such as Fluka Cat 71736 1 100 in pure water ABN O PinPoint solution is used only for the protocol listed under section B below 2 Deparaffinize Tissue Sections 1 Immerse slides in xylene for 15 minutes 2 Transfer slides to a second xylene bath for 15 minutes 3 Immerse slides in a pure ethanol bath for 5 minutes 4 Repeat the ethanol bath twice more for a total of 3 ethanol baths 5 Allow slides to dry at room temperature 3 Sample Preparation Using SDS 1 Pipet 50 ul of 0 1 SDS onto the dried section Scrape up section using a pipet tip and transfer as completely as possible to a 1 7 mL tube O It is difficult to remove all of the section using a small aliquot of SDS The following two washing steps are done to remove as much residual tissue section as possible 2 Add 100 ul of 0 1 SDS to the slide and scrape up and remove any remaining section 3 Rinse the slide with an additional 100 ul aliquot of 0 1
9. k through the channel 5 J Step 4 With a pipettor at the Exit Port remove the eluate into a clean tube 10 Application Notes BCSI Product Matrix i BCSI is developing new applications and improved protocols on an ongoing basis The most current revision of this document will always be available through our website www bloodcellstorage com Applications Ordering Info User Reference Lysis BCSI Kit Buffer Protease Carrier Total NA from Plasma SNAP 02 002R 0 GT SNAP Protease 1 N A Total NA from FFPE Tissue SNAP 02 004R 0 GT SNAP Protease 2 N A Total NA from E coli SNAP 02 005R 0 GT SNAP Protease 2 N A Viral RNA from Plasma SNAP 02 006R 0 GT ND SNAP Protease 1 SNAP Carrier 1 Total Nucleic Acids from Plasma 1 Required Reagents 1 BCSI SNAP GT Lysis 2 BCSI SNAP Protease 1 2 Sample Preparation 1 Aliquot 0 5 mL of Cell Free Plasma into 1 7 mL tubes 2 Add 1 drop BCSI SNAP Protease 1 Diluted to 5 mg mL as instructed 200 ug 3 Incubate at room temperature or higher for 30 minutes 4 Add 1 mL SNAP GT Lysis buffer and mix well 3 Rapid Procedure from 0 5 mL Plasma 1 Load the sample into a SNAP card Let the sample sit for at least 30 minutes 2 Wash and dry as directed 3 Elute using 100 ul Elution Buffer 4 High Yield Procedure A O The high yield procedure yields 2 3 times more nucleic acid using 2 mL of plasma than the rapid protocol Higher
10. l pure ethanol and mix well With a 1 ml pipetor fixed with a wide orifice pipet tip load on the entire sample through the entry port Try not to introduce excessive bubbles Let sit at least 30 minutes or up to 2 hours Longer binding times may result in increased capture Wipe any residual sample or sample deposits from the surface of the card Wash and dry the cards per the above general instructions 15 Appendix A Risk and Safety Phrases Component Hazard Contents Hazard Code Risk Phrases Satety Phrases SNAP GT Lysis Guanidine thiocyanate 20 21 22 32 52 53 13 22 61 SNAP Wash 1 E i Guanidine hydrochloride 20 21 22 32 52 53 13 22 61 SNAP Protease 1 36 37 38 42 22 24 26 36 37 39 SNAP Washi 2 Proclin 150 34 43 26 36 37 39 45 Concentrate SNAP Elution Sodium Azide T N 28 32 50 53 28 45 60 61 SNAP Protease 2 36 37 38 42 22 24 26 36 37 39 Hazard Codes Xoana Harmful Conania a Corrosive Niciraritarc nias Dangerous for the environment A Aa Very Toxic Risk Phrases Ll AT Harmful if Swallowed PO Very Toxic if swallowed Vd Contact with acids liberates very toxic gas A O Causes burns TA Risk of serious damage to eyes ATEREA ETT May cause sensitization by inhalation Acid ds May cause sensitization by skin contact LO iii Harmful by inhalation in contact with skin and if swallowed 36 37 38 wi scciceatiliees Irritating to eyes respiratory system and skin SH BB RETE Irritating to respiratory syste
11. m and skin 0 58 as aiaiai Very Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment DUI didas Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment DL alt Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Safety Phrases E ENS PTE Keep away from food drink and animal feeding stuffs E iS Do not breathe dust Ci dad Avoid contact with skin DU aiii In case of contact with eyes rinse immediately with plenty of water and seek medical advice After contact with skin wash immediately with plenty of water In case of accident or if you feel unwell seek medical advice immediately show label where possible This material and or its container must be disposed of as hazardous waste Avoid release to the environment Refer to special instructions Safety Data Sheet Wear suitable protective clothing and eye face protection Wear suitable protective clothing and gloves Wear suitable protective clothing gloves and eye face protection 16
12. ncentrate ss 60 mL Elution Buffer 50 mL Supplemental Reagents optional SNAP Protease 1 SNAP Protease 2 SNAP Carrier 1 Storage SNAP products are stored at room temperature Protect all SNAP Products from freezing Refer to bottles for storage instructions for supplemental reagents Equipment and Reagents Supplied by the User Pipettes Ethanol 95 100 ACS USP Grade Sodium dodecyl sulfate solution Fluka Cat 71736 or equivalent Xylene 1 7mL microcentrifuge tubes VWR 100 1000 ul Wide Orifice Pipet Tips Cat 89049 168 or equivalent to fit 1000 ul pipette NOTE It is critical to have tips that fit the entry port of the SNAP Card Vortex Mixer Microcentrifuge Disposable Gloves Graduated Cylinder Vacuum Desiccator for manual operation only A CAUTION Do not use denatured alcohol which contains other substances such as methanol or methylethylketone Product Use Limitations The BCSI SNAP product line consists of a set of general purpose reagents intended for research use only and accessory general research use equipment This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of BCSI products adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Quality Control Application is for General Research Use Techni
13. ntry Port A E A o spaces BCSI Nucleic Acid Extraction Card A Step 3 Introduce Elution Buffer Step 6 Draw elution bubble through the Exit Port towards Exit Port 8 lt gt als crane apne ot aa Step 4 Move the Bubble of Step 7 Remove elution bubble 9 Elution Buffer towards the Entry Port through the Exit Port Remove Sample Through Entry Port En no y Wash lol f 2 gt ER a a Ne a gt aa 7 gt Wash Out SS Eee id y Remo gt as ra Step 2 Wash and Dry Card Step 5 Draw the elution bubble back towards the Exit Port Automated Card Washing 1 Prepare the wash station SNAP R for use according to its user manual Be sure to follow all instructions for maintaining equipment cleanliness 2 Load the card onto the card washing station following the procedure described in the equipment documentation Program the wash station using the parameters listed below Start the program The station will wash the channel on the card thoroughly dry away residual ethanol and if desired introduce the elution buffer to the card Wash Parameters Washi kaaa 2 Washes of 1 mL each Wash2 y aaa 6 Washes of 1 mL each Drying Time 3 Minutes Elution Volume 100 200 ul O manual elution is to be performed ensure the wash station program does not call for elution to be introduced by the machine Automated C
14. option See www bloodcellstorage com to learn more about the SNAP M option Manual Elution Elution is to be performed on cards after washing and drying are complete 1 Aliquot 100 200 pl of Elution Buffer to a fresh clean tube 2 With a fresh 1 mL pipet tip transfer the Elution Buffer to the exit port 3 Move the Bubble of Elution Buffer from the exit port to the entry port slowly The goal is to assure that the Elution Buffer is in good contact with the entire S channel surface Occasionally the Bubble will break contact with the side of the channel and the bubble will not move Lightly tap the card on its side to re establish contact and complete the elution 4 Allow the bubble to sit at entry point for 10 20 minutes Move the bubble slowly from the entry port to the exit port and remove the eluate through the exit port to a clean tube When introducing Elution Buffer into a dry card it is normal for some of the liquid to be retained by the card Depending on the properties of the sample expected volume recovered can range between 50 75 of volume introduced 5 Repeating the elution process may allow additional recovery of nucleic acids O Repeated sweeping of the channel with the elution bubble does not significantly increase yield A second elution should be performed with fresh Elution Buffer BCSI Nucleic Acid Extraction System Guide Removal of Eluate With Manual Elution A Exit Port So i E
15. rm the solution to redissolve completely SNAP Wash 1 The Wash 1 bottle contains 60 mL of wash 1 concentrate Add 30 mL of ethanol Mix well NOTE Improper preparation of Wash 1 can result in failed extraction SNAP Wash 2 The Wash 2 bottle contains 60 mL of wash 2 concentrate Add 140 mL of ethanol Mix well NOTE Improper preparation of Wash 2 can result in failed extraction SNAP Elution Buffer The Elution buffer is shipped ready to use and is confirmed to be RNase free CAUTION Elution Buffer contains Sodium Azide Additional Components Available from BCSI SNAP Protease 1 amp SNAP Protease 2 A protease step during sample prep may increase the yield BCSI provides two protease options SNAP Protease 1 and SNAP Protease 2 Both are available in a convenient dropper To prepare protease for use remove from freezer thirty 30 minutes prior to reconstitution To reconstitute remove the cap from the bottle and the dropper attachment Add 2 mL of elution buffer Mix gently and then let the bottle sit for a few minutes to fully resuspend the protein Then gently mix again Reconstituted protease is best stored at 20 C for up to one year The protease loses about 20 of activity when stored refrigerated and greater than 50 activity when stored at room temperature for 1 week The reconstituted protease can undergo at least 12 freeze thaw cycles without significant loss of activity SNAP Carrier 1 Carrier RNA is available from BCSI SN
16. yields can be obtained by increasing the number of plasma loading steps or increasing the binding time Prepare four separate tubes of sample for each card Load the first sample onto a card and let sit for at least 30 minutes to allow nucleic acid adsorption Remove the sample and discard in biological waste Remove the sample and repeat with the remaining 2 sample tubes for a total of 4 steps Wash and dry as directed in the Instructions for Use 1 2 3 4 Load on the second sample Let sit for at least 30 minutes 5 6 7 Elute using 100 ul Elution Buffer 11 4 High Yield Procedure B O This procedure yields at least 2 times more nucleic acid than the rapid protocol It uses 2 extraction cards and 1 mL of plasma and a serial elution procedure in which the eluate from the first card is used to elute the second card 1 Prepare two separate tubes of sample each using 0 5 mL sample 2 Load the samples onto each of two cards and let sit for a minimum of 2 hours to allow nucleic acid adsorption Overnight incubation at room temperature gives the best yield 3 Wash and dry the cards as directed by the Instructions for Use 4 To serially elute the cards carefully load 150 ul of elution buffer on the first card Slowly move the buffer to the opposite port and let the buffer sit on the card for 10 20 minutes Then slowly bring the elution buffer back across the channel and remove 5 Load the retrieve
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