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        Cloning of Taq polymerase-amplified PCR products

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        1204

        实验原理

         

        The plasmid vector (pCR®II-TOPO® or pCR®2.1-TOPO®) is supplied linearized with:

        Single 3´-thymidine (T) overhangs for TA Cloning®

        Topoisomerase I covalently bound to the vector (referred to as "activated" vector)

        Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

        Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5´-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3´ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5´ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994).

        实验步骤

         

        1.        Producing PCR Products for TOPO® TA Cloning

        1)        Set up the following 50-µl PCR reaction using Taq DNA polymerase. Use less DNA if you are using plasmid DNA as a template and more DNA if you are using genomic DNA as a template. Use the cycling parameters suitable for your primers and template. Be sure to include a 7 to 30 minute extension at 72°C after the last cycle to ensure that all PCR products are full length and 3´ adenylated.

                                                                       Table 1

        DNA Template

        10-100 ng

        10X PCR Buffer

        5 µl

        50 mM dNTPs

        0.5 µl

        Primers (100-200 ng each)

        1 µM each

        Water

        add to a final volume of 49 µl

        Taq Polymerase (1 unit/µl)

        1 µl

        Total Volume

        50 µl

         

        2)        Check the PCR product by agarose gel electrophoresis. You should see a single, discrete band. If you do not see a single band, refer to the Note below.

        2.        Optional Protocol with Platinum® Taq DNA Polymerase High Fidelity

        1)        Add the following components to a autoclaved microcentrifuge tube at either ambient temperature, or on ice:

        2)        Mix contents of the tubes and overlay with mineral or silicone oil, if necessary.

        3)        Cap the tubes and centrifuge briefly to collect the contents.

        4)        Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min to completely denature the template and activate the enzyme.  Do not denaturate for more than 30 s if target is greater than 12 kb.

        5)        Perform 25-35 cycles of PCR amplification as follows:

                          Denature       94°C for 15–30 s

                           Anneal          55°C for 15–30 s

                           Extend           68°C for 1 min per kb

        6)        Maintain the reaction at 4°C after cycling.  The samples can be stored at -20°C until use.

        7)        Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining.  Use appropriate molecular weight standards.

        3.        Purifying PCR Products

        1)        Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel.

        2)        Note:  Do not use TBE. Borate interferes with the NaI step (Step 2).

        3)        Cut out the gel slice containing the PCR product and melt it at 65°C in 2 volumes of 6 M NaI.

        4)        Add 1.5 volumes Binding Buffer (provided in the S.N.A.P. ™ MiniPrep Kit).

        5)        Load solution (no more than 1 ml at a time) from Step 3 onto a S.N.A.P. ™ column. Centrifuge 1 minute at full speed in a microcentrifuge and discard the supernatant.

        6)        If you have solution remaining from Step 3, repeat Step 4.

        7)        Add 900 µl of the Final Wash Buffer (provided in the S.N.A.P. ™ MiniPrep Kit).

        8)        Centrifuge 1 minute at full speed in a microcentrifuge and discard the supernatant. Repeat.

        9)        Elute the purified PCR product in 40 µl of TE or water. Use 4 µl for the TOPO® Cloning reaction.

        4.        Setting Up the TOPO® Cloning Reaction

        1)        Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C).

        Note:  For most applications, 5 minutes will yield plenty of colonies for analysis. Depending on your needs, the length of the TOPO®Cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO® Cloning a pool of PCR products, increasing the reaction time will yield more colonies.

        2)        Place the reaction on ice and proceed to General Guidelines for Transforming Competent Cellsnext page.

        Note:  You may store the TOPO® Cloning reaction at -20°C overnight.

        5.        Analyzing the Transformants

        1)        Culture 10 white or light blue colonies overnight in LB medium containing 50 µg/ml ampicillin or 50 µg/ml kanamycin.

        2)        Note:  If you transformed One Shot® Mach1™-T1R competent E. coli, you may inoculate overnight-grown colonies and culture them for 4 hours in prewarmed LB medium containing 50 µg/ml ampicillin or 50 µg/ml kanamycin before isolating plasmid. For optimal results, we recommend inoculating as much of a single colony as possible.

        3)        Isolate plasmid DNA using your method of choice. If you need ultra-pure plasmid DNA for automated or manual sequencing, we recommend the S.N.A.P.™ MiniPrep Kit (Catalog no. K1900-01) or the S.N.A.P.™ MidiPrep Kit (Catalog no. K1910-01).

        4)        Analyze the plasmids by restriction analysis to confirm the presence and correct orientation of the insert. Use a restriction enzyme or a combination of enzymes that cut once in the vector and once in the insert.

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