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Cloning of Taq polymerase-amplified PCR products

互联网2013-11-13

1076

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.

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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