SMARTer RACE 5'3' Kit Protocol-At-A-Glance_032514


    SMARTer® RACE 5’3’ Kit ProtocolAtAGlance
    (032514) wwwclontechcom
    Clontech Laboratories Inc A Takara Bio Company
    Page 1 of 6


    Please read the User Manual for the SMARTer® RACE 5’3’ Kit (Cat Nos 634858 634859) before using this Protocol
    AtAGlance This abbreviated protocol is provided for your convenience but is not intended for firsttime users
    I Primer Design (Section IV of the User Manual)
    GeneSpecific Primers (GSPs) should
     be 23–28 nt to ensure specific annealing
     be 50–70 GC
     have a Tm ≥65°C best results are obtained if Tm >70°C which enables the use of touchdown PCR (Tm should
    be calculated based upon the 3’ (genespecific) end of the primer NOT the entire primer)
     not be complementary to the 3’end of the Universal Primer Mix
    Long primer 5’–TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT–3'
    Short primer 5’–CTAATACGACTCACTATAGGGC–3’
     be specific to your gene of interest
     both have 15 bp overlaps with the vector at their 5’ ends to facilitate InFusion® cloning (ie add the
    sequence GATTACGCCAAGCTT to the 5’ ends of both GSPs’ sequences)
    II Generating RACEReady cDNA (Section V of the User Manual)
    1 Prepare enough of the following Buffer Mix for all of the 5’ and 3’RACEReady cDNA synthesis reactions
    plus 1 extra reaction to ensure sufficient volume Mix the following reagents and spin briefly in a
    microcentrifuge then set aside at room temperature until Step 6
    40 µl 5X FirstStrand Buffer
    05 µl DTT (100 mM)
    10 µl dNTPs (20 mM)
    55 µl Total Volume
    2 Combine the following reagents in separate microcentrifuge tubes
    For preparation of
    5’RACEReady cDNA For preparation of
    3’RACEReady cDNA
    10–10 µl RNA* 10–11 µl RNA*
    10 µl 5’CDS Primer A 10 µl 3’CDS Primer A
    0–9 µl Sterile H2O 0–10 µl Sterile H2O
    11 µl Total Volume 12 µl Total Volume
    *For the control reactions use 1 µl of Control Mouse Heart Total RNA (1 µgµl)
    3 Mix contents and spin the tubes briefly in a microcentrifuge
    4 Incubate tubes at 72°C for 3 minutes then cool the tubes to 42°C for 2 minutes After cooling spin the tubes
    briefly for 10 seconds at 14000 x g to collect the contents at the bottom
    NOTE This step can be performed in a thermal cycler While the tubes are incubating you can prepare the
    Master Mix in Step 6
    5 To just the 5’RACE cDNA synthesis reaction(s) add 1 µl of the SMARTer II A Oligonucleotide per
    reaction
    SMARTer® RACE 5’3’ Kit ProtocolAtAGlance
    (032514) wwwclontechcom
    Clontech Laboratories Inc A Takara Bio Company
    Page 2 of 6


    6 Prepare enough of the following Master Mix for all 5’ and 3’RACEReady cDNA synthesis reactions Mix
    these reagents at room temperatures in the following order
    55 µl Buffer Mix from Step 1
    05 µl RNase Inhibitor (40 Uµl)
    20 µl SMARTScribe™ Reverse Transcriptase (100 U)
    80 µl Total Volume
    7 Add 8 µl of the Master Mix from Step 6 to the denatured RNA from Step 4 (3’RACE cDNA) and Step 5 (5’
    RACE cDNA) for a total volume of 20 µl per cDNA synthesis reaction
    8 Mix the contents of the tubes by gently pipetting and spin the tubes briefly to collect the contents at the bottom
    9 Incubate the tubes at 42°C for 90 minutes in an air incubator or a hotlid thermal cycler
    10 Heat tubes at 70°C for 10 minutes
    11 Dilute the firststrand cDNA synthesis reaction product with TricineEDTA Buffer
     Add 10 µl if you started with <200 ng of total RNA
     Add 90 µl if you started with >200 ng of total RNA
     Add 240 µl if you started with poly A+ RNA

    12 You now have 3’ and 5’RACEReady cDNA samples Samples can be stored at –20°C for up to three months
    III Rapid Amplification of cDNA Ends (RACE) (Section VI of the User Manual)
    This procedure describes the 5’RACE and 3’RACE PCR reactions that generate the 5’ and 3’ cDNA fragments
    We recommend that you also perform positive control 5’ and 3’RACE using the TFR primers and UPM
    Although the Universal Primer Short (UPM short) is provided nested PCR is generally not necessary in
    SMARTer RACE reactions
    1 Prepare enough PCR Master Mix for all of the PCR reactions plus one extra reaction to ensure sufficient
    volume The same Master Mix can be used for both 5’ and 3’RACE reactions For each 50 µl PCR reaction
    mix the following reagents
    155 µl PCRGrade H2O
    250 µl 2X SeqAmp™ Buffer
    10 µl SeqAmp DNA Polymerase
    415 µl Total Volume


    SMARTer® RACE 5’3’ Kit ProtocolAtAGlance
    (032514) wwwclontechcom
    Clontech Laboratories Inc A Takara Bio Company
    Page 3 of 6


    2 Prepare PCR reactions as shown below in Table 1 Add the components to 05 ml PCR tubes in the order
    shown and mix gently
    Table 1 Setting up 5' and 3'RACE PCR Reactions
    Component 5’ or 3’RACE
    Sample
    UPM only
    (– control)
    GSP only
    (– control)
    5’ or 3’RACEReady cDNA
    (experimental) 25 µl 25 µl 25 µl
    10X UPM 5 µl 5 µl —
    5’ or 3’ GSP (10 µM) 1 µl — 1 µl
    H2O — 1 µl 5 µl
    Master Mix (Step 1) 415 µl 415 µl 415 µl
    Total Volume 50 µl 50 µl 50 µl

    3 Commence thermal cycling using one of the following PCR programs (both programs 1 and 2 work with the
    positive control 5’ and 3’RACE TFR and UPM Primers) Be sure to choose the correct number of cycles (as
    noted) based on whether you started with poly A+ or total RNA
    NOTES ON CYCLING You may need to determine the optimal cycling parameters for your gene
    empirically because the number of cycles necessary depends on the abundance of the target transcript The
    optimal extension time depends on the length of the desired amplicon For 02–2 kb amplicons we typically
    extend for 2 minutes for 2–4 kb amplicons we extend for 3 minutes and for 5–10 kb amplicons we extend
    for up to 10 minutes

    NOTE The Tm should be calculated based upon the 3’ (genespecific) end of the primer and NOT the entire
    primer
    Program 1 (touchdown PCR—preferred use if GSP Tm >70°C)
     5 cycles
    94°C 30 sec
    72°C 3 min*
     5 cycles
    94°C 30 sec
    70°C 30 sec
    72°C 3 min*
     20 cycles (Poly A+ RNA) OR 25 cycles (Total RNA)
    94°C 30 sec
    68°C 30 sec
    72°C 3 min*
    *If fragments >3 kb are expected add 1 minute for each additional 1 kb

    SMARTer® RACE 5’3’ Kit ProtocolAtAGlance
    (032514) wwwclontechcom
    Clontech Laboratories Inc A Takara Bio Company
    Page 4 of 6


    Program 2 (use if GSP Tm 60–70°C)
     20 cycles (Poly A+ RNA) OR 25 cycles (Total RNA)
    94°C 30 sec
    68°C 30 sec
    72°C 3 min*
    *If fragments >3 kb are expected add 1 minute for each additional 1 kb
    IV Characterization of RACE Products (Section VII of the User Manual)
    A Gel Extraction with the NucleoSpin Gel and PCR CleanUp Kit
    For more details on the included NucleoSpin Gel and PCR CleanUp Kit please download its User
    Manual from our website at wwwclontechcommanuals
    Before you start Add 24 ml of 96–100 ethanol to Wash Buffer NT3 Mark the label of the bottle to
    indicate that ethanol was added Wash Buffer NT3 is stable at room temperature (18–25°C) for at least
    one year
    1 Electrophorese your RACE DNA sample on an agaroseEtBr gel We recommend using a buffer
    system containing either TAE (40 mM Trisacetate [pH 8] 1 mM EDTA) or TBE (45 mM Trisborate
    [pH 8] 1 mM EDTA)
    2 Locate the position of your fragment under UV light Use a clean scalpel or razor blade to excise the
    DNA fragment of interest Cut close to the fragment to minimize the surrounding agarose Estimate
    the amount of DNA present in the gel slice
    NOTE Minimize UV exposure time to avoid damaging the DNA
    3 Measure the weight of the gel slice and transfer it to a clean 15 ml microcentrifuge tube
    4 For each 100 mg of agarose add 200 µl Buffer NTI
    5 Incubate the sample for 5–10 minutes at 50°C Vortex every 2–3 minutes until the gel slice is
    completely dissolved
    6 Place a NucleoSpin Gel and PCR CleanUp Column into a Collection Tube (2 ml) and load up to
    700 µl of sample Centrifuge for 30 seconds at 11000 x g Discard flowthrough and place the
    column back into the collection tube Load remaining sample if necessary and repeat the
    centrifugation step
    7 Add 700 µl Buffer NT3 to the column Centrifuge for 30 seconds at 11000 x g Discard flowthrough
    and place the column back into the collection tube
    8 Centrifuge for 1 minute at 11000 x g to remove Buffer NT3 completely Make sure the spin column
    does not come in contact with the flowthrough while removing it from the centrifuge and collection
    tube
    NOTE Residual ethanol from Buffer NT3 might inhibit enzymatic reactions Total removal of
    ethanol can be achieved by incubating the columns for 2–5 minutes at 70°C prior to elution (Step 9)
    SMARTer® RACE 5’3’ Kit ProtocolAtAGlance
    (032514) wwwclontechcom
    Clontech Laboratories Inc A Takara Bio Company
    Page 5 of 6


    9 Place the column into a new 15 ml microcentrifuge tube (not provided) Add 15–30 µl Buffer NE
    and incubate at room temperature (18–25°C) for 1 minute Centrifuge for 1 minute at 11000 x g to
    elute DNA
    NOTE DNA recovery of larger fragments (>1000 bp) can be increased by multiple elution steps
    with fresh buffer heating to 70°C and incubation for 5 minutes
    B InFusion Cloning of RACE Products
    For more details on the included InFusion HD Cloning Kit please download its User Manual from our
    website at wwwclontechcommanuals
    1 Combine
    1 µl Lineareized pRACE vector (provided with SMARTer RACE 5’3’ Kit Components)
    7 µl Gelpurified RACE product
    2 µl InFusion HD Master Mix
    10 µl Total Volume
    2 Incubate for 15 minutes at 50°C and transfer to ice
    3 Follow the protocol provided with your Stellar™ Competent Cells to transform the cells with 25 µl
    of the InFusion reaction mixture
    IMPORTANT DO NOT add more than 5 µl of the reaction to 50 µl of competent cells More is not
    better Using too much of the reaction mixture inhibits the transformation
    4 Place 1100–15 of each transformation reaction into separate tubes and bring the volume to 100 µl
    with SOC medium Spread each diluted transformation on a separate LB plate containing 100 µgml
    of ampicillin
    5 Centrifuge the remainder of each transformation at 6000 rpm for 5 minutes Discard the supernatant
    and resuspend each pellet in 100 µl fresh SOC medium Spread each sample on a separate LB plate
    containing the appropriate antibiotic Incubate all of the plates overnight at 37°C
    6 The next day pick individual isolated colonies from each experimental plate Isolate plasmid DNA
    using a standard method of your choice (eg miniprep) To determine the presence of your RACE
    insert analyze the DNA by PCR screening (with your GSPs) or restriction digest (with EcoRI and
    HindIII which flank the cloning site)
    NOTE For 5’RACE products we recommend picking at least 8–10 different independent clones in
    order to obtain the maximum amount of sequence at the 5’end
    C Sequencing RACE Products
    Once you have identified the clones containing the largest genespecific inserts obtain as much sequence
    data as you can Ideally you will be able to sequence the entire open reading frame as well as the 5’ and
    3’ untranslated regions

    SMARTer® RACE 5’3’ Kit ProtocolAtAGlance
    (032514) wwwclontechcom
    Clontech Laboratories Inc A Takara Bio Company
    Page 6 of 6


    NOTE The provided pRACE vector is a pUC19based vector and is compatible with M13 sequencing
    primers for characterization of your cloned insert(s) Because InFusion cloning is directional you can
    preferentially use the M13F primer to sequence into the UPM end and the M13R primer to sequence into
    the genespecific end

    The UPM contains a T7 priming site which can be used for Sanger sequencing but we recommend using
    M13 primers to get full clean reads of your experimental sequence The T7 priming sites are too close to
    the 5’ and 3’cloning sites to ensure complete coverage in the sequencing trace
    Options for generating fulllength cDNA
    After the RACE products have been characterized by partial or complete sequencing you can generate
    the fulllength cDNA by one of two methods
     By long distance PCR (LD PCR) using primers designed from the extreme 5’ and 3’ ends of your
    cDNA and the 5’RACEReady cDNA as a template
     By cloning overlapping 5’ and 3’RACE fragments using a restriction site in the overlapping region
    (if available) If no suitable restriction sites are available you can alternately design new GSPs
    suitable for multifragment InFusion cloning
    NOTE Details on multifragment InFusion cloning can be found in our tech note InFusion Multiple
    Fragment Cloning

    Contact Us
    Customer ServiceOrdering Technical Support
    tel 8006622566 (tollfree) tel 8006622566 (tollfree)
    fax 8004241350 (tollfree) fax 8004241350 (tollfree)
    web wwwclontechcom web wwwclontechcom
    email orders@clontechcom email tech@clontechcom

    Notice to Purchaser
    Clontech® products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro
    diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture
    commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories Inc
    Your use of this product is also subject to compliance with any applicable licensing requirements described on the product’s web page at httpwwwclontechcom It is
    your responsibility to review understand and adhere to any restrictions imposed by such statements
    Clontech the Clontech logo InFusion SeqAmp SMARTer SMARTScribe and Stellar are trademarks of Clontech Laboratories Inc All other marks are the property
    of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company ©2014 Clontech Laboratories Inc
    This document has been reviewed and approved by the Clontech Quality Assurance Department

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