Purification of Blunt-Ended Plasmid DNA
We routinely use the Wizard DNA Clean-up System to purify DNA, and the major steps are listed below. When using the Wizard DNA Clean-up kit, it is important to elute the DNA from the resin with water. EDTA in the TE buffer will chelate Mg++, thus reducing the working concentration of Mg++ in the next enzyme incubation step. The phenol/ chloroform extraction and ethanol precipitation method also works well.
1. Add 1 ml of well mixed Wizard DNA Clean-up resin to the tube containing the enzyme digestion products, and mix by inverting for 1 min.
2. Attach a 3 ml disposable syringe barrel (without the plunger) to the minicolumn provided, and insert the tip of the minicolumn/syringe barrel assembly into a vacuum manifold.
3. Pipet the DNA/resin mix into the syringe barrel. Apply the vacuum to draw the mix into the minicolumn.
4. Pipet 2 ml of 80% isopropanol into the syringe barrel, and apply the vacuum to draw the solution through the minicolumn.
5. Remove the syringe barrel and transfer the minicolumn to a 1.5 ml centrifuge tube. Centrifuge the minicolumn for 2 min at 12,000 x g.
6. Attach the minicolumn to the vacuum source, and apply the vacuum for another 2 min.
7. Transfer the minicolumn to a new 1.5 ml microcentrifuge tube, add 50 µl of water (pre-warmed to 65°C) to the minicolumn, and wait for 1 min. Centrifuge the minicolumn for 20 s at 12,000 x g. The DNA is collected in the microcentrifuge tube.
Addition of T-overhangs to the Blunt-ended Plasmid Vector
Two widely used methods are described. The first method employs terminal deoxynucleotidyl-transferase (TdT) and dideoxythymidine triphosphate (ddTTP) to add a ddT residue to the blunt-ended plasmid, which ensures the addition of only a single T residue (10). In the second method, Taq polymerase and dTTP are used to add a 3'-T to the blunt-ended plasmid (11 ). Since Taq polymerase adds the 3'-T residues inefficiently, only a portion of the blunt-ended plasmids are T-tailed at both ends, whereas the remainder are T-Tailed only at one end or do not have T-tails at all. Therefore, the vectors without T-overhangs are eliminated from the T-vectors by gel purification after self-ligation and/or concatemerization by incubation with T4 DNA ligase (16 ). Unlike Taq polymerase, terminal transferase adds ddT to 3'-ends very efficiently. Therefore consistent and high cloning efficiency is obtained using T-vectors prepared by TdT, even without ligation and gel purification steps. However, the efficiency can be further increased by incubation with T4 DNA ligase, followed by gel separation.
A. Terminal Transferase Method This method originated from Holton and Graham (10), with minor modifications in our laboratory. Terminal transferase from Boehringer Mannheim is strongly recommended. TdT from other sources may not work as well. The final concentration of ddTTP in the reaction is 20 µM; higher concentrations are unnecessary.
1. Pipet 46 µl of purified blunt-ended plasmid DNA (from step 7 of "Purification of Blunt-ended Plasmid DNA") into a 1.5 ml centrifuge tube (if the volume is less, add sterile water to bring the volume to 46 µl), then mix
the following reagents:
Purified Blunted plasmid DNA 46 µl
5 X TdT buffer 15 µl
25 mM CoCl2 7.5 µl
1 mM ddTTP 1.5 µl
Terminal Transferase (25 U/µl) 5 µl
Mix by gentle vortexing and centrifuge briefly.
2. Incubate at 37°C for 1.5 h, then proceed to the "Purification of the T-tailed Vectors" (see below).
B. Taq Polymerase Method
1. Adjust the volume of the purified blunted plasmid DNA (from step 7 of "Purification of Blunt-ended Plasmid DNA") to 83 µl by adding sterile water, then mix the following reagents:
Purified blunted plasmid DNA 83 µl 10 x PCR buffer (MgCl2 free) 10 µl 50 mM MgCl2 5 µl 100 mM dTTP 1 µl Taq polymerase (5 U/µl) 1 µl
Mix well and centrifuge briefly.
2. Incubate at 72°C for 2 h, then proceed to the "Purification of the T-tailed Vectors" (see below).
Purification of the T-tailed Vectors
The T-tailed plasmid DNA solution obtained by any one of the methods described above is purified by employing the Wizard DNA Clean-up System as described above or phenol/chloroform extraction and ethanol precipitation.
Ligation of Vectors without T-tails If you use the Taq polymerase method to make the T-vectors, removal of the vectors without T-tails is necessary and improves the cloning efficiency up to 80% (16). If the terminal transferase method and a plasmid without blue/ white selection (for example pcDNA3) is used to make T-vectors, the cloning efficiency is about 70%, without further purification. Elimination of the vectors without T-overhangs by gel separation, following incubation with T4 DNA ligase, increases the cloning efficiency to 90%. If you use the terminal transferase method to make T-vectors, and the plasmid has blue/white selection capabilities, ligation and gel purification may not be necessary (Go directly to "Quantification and Storage of the Purified T-vector" below if time savings is desired).
1. To a 1.5 ml microcentrifuge tube, add T-tailed plasmid DNA (as purified above) 44 µl 10x ligase buffer 5 µl T4 DNA ligase (3 U/µl) 1 µl
Mix well and centrifuge briefly.
2. Incubate at 16°C overnight.
Separation and Recovery of T-vectors on an Agarose Gel
T-vectors are separated from the self-ligated and concatemerized plasmid DNA on a 1% agarose gel. The T-vector band is cut out and recovered using the Wizard PCR Preps Kit.
1. Separate the ligation mixture (from above) on a 1% low-melting agarose gel with 0.5 µg/ml of ethidium bromide. Run the gel in TAE buffer at 5 volts/cm.
2. Visualize the DNA bands with a hand-held long wave (365 nm) UV light. Excise the T-vector band with a sterile scalpel. Transfer the gel slice to a 2 ml microcentrifuge tube.
3. Incubate the gel slice in a 65°C water bath for 5 min, or until melted.
4. Add 1 ml of Wizard PCR preparation resin (which has been thoroughly mixed) to the melted agarose slice. Mix thoroughly by inverting for 1 min.
5. Follow steps 2 to 7 of the " Purification of Blunt-Ended Plasmid DNA" to complete the purification.
Quantification and Storage of the Purified T-vector
The concentration of the purified T-vector or other DNA can be conveniently estimated by running an aliquot of the DNA side by side with a known amount of a DNA standard in a 1% agarose gel, and then comparing the relative brightnesses of the bands. The electrophoresis is run at a higher than normal voltage for a short time (usually 10-15 min) to allow the DNA to more efficiently migrate into the gel. Standard loading buffer contains bromphenol blue and xylene cyanol which may interfere with the visualization of band brightness. Therefore, it is strongly suggested that a 6 X loading buffer without these dyes be used for quantification purposes. The T-vectors are aliquoted and stored at -20°C to reduce multiple freeze-thaw cycles.
1. Run 1 µl of the purified T-vector and a known amount of standard DNA side by side (use loading buffer without any dyes) in a 1% agarose gel at 10 volts/cm for 10-15 min.
2. Compare the band brightnesses to determine the concentration of the T-vector.
3. Aliquot the T-vector to several tubes and store them at -20°C.
5. Add 2 µl of 0.5 M EDTA to stop the reaction. Then proceed to " Separation and Recovery of DNA Fragments from an Agarose Gel" .
B. DNA Fragments Amplified by PCR
Large amounts of DNA can be obtained by PCR. Many variations and applications of this technique, such as RT-PCR (reverse transcription PCR), 5'-RACE (rapid amplification of 5'-cDNA ends), and XL-PCR (extra long PCR), have been developed to serve various needs. A successful PCR amplification starts with the selection of appropriate primer pairs and then optimization of the PCR conditions (such as annealing temperature, extension time, and Mg++ concentration). Several thermal stable enzymes such as Taq, Vent, pfu, and Tth polymerases, are available. General PCR references should be consulted to select the enzyme best suited to your needs (17, 18). After successful PCR amplification, proceed to the next stage to clone the amplified DNA product.
Separation and Recovery of DNA Fragments from an Agarose Gel
The desired DNA fragments from restriction digestion are separated from other DNA fragments by agarose gel electrophoresis and the desired bands are cut out and recovered. PCR products consisting of a single specific band amplified by Taq polymerase can be directly ligated to T-vectors. However, the removal of the impurities such as primers and trace amounts of nonspecific products by gel separation or the use of direct DNA clean-up systems increases the percentage of colonies containing the correct inserts. If you use a thermo-stable DNA polymerase which does not preferentially add a 3'-A at the ends of the PCR products, removal of that enzyme by either gel separation or use of a direct DNA clean-up system is essential. When two or more desired PCR products are amplified in the same PCR reaction, or when the nonspecific amplification products are abundant enough to be visualized on a gel, it is necessary to purify the desired DNA fragments by gel electrophoresis (to avoid screening the positive colonies later by more complicated hybridization procedures). Purify and recover the DNA from agarose gels as described in the "Separation and Recovery of T-vectors on an Agarose Gel" section of protocol 1. In addition, the concentration of the purified DNA is estimated as described in protocol 1.
A-tailing of the Purified DNA using Taq Polymerase In the presence of all four dNTPs, Taq polymerase will first fill in the 3'-recessed bases of the DNA fragment with 5'-overhangs and then preferentially add A's to the 3'-ends. Alternatively, the Taq enzyme will add an A directly to the 3'-ends of a blunt-ended DNA fragment. In the case of PCR products or blunt-ended fragments, only dATP is necessary in the incubation mixture. However, a mixture of dNTPs works well in all cases, since Taq polymerase preferentially adds an A to the 3'-ends in the presence of all four dNTPs.
Before starting this step, calculate the concentration of the DNA fragment needed in a 10 µl ligation reaction to give a 1:2 molar ratio of the T-vector to the inserts. Since no more than 2 µl of A-tailed DNA is used in the ligation reaction, the final concentration of the DNA fragment in the A-tailing reaction mixture should be adjusted so that the amount of DNA fragment required for ligation will be contained in a volume of 2 µl or less. Should the DNA concentration be too low, you may concentrate the DNA before the Taq polymerase incubation step.
1. To a 0.5 ml sterile centrifuge tube, add: Purified DNA fragment 7.7 µl 10 X PCR buffer, Mg++- free 1 µl 25 mM MgCl2 1 µl dNTP mix, 10 mM each 0.2 µl Taq polymerase (5 U/µl) 0.1 µl
Mix well and centrifuge briefly.
2. Incubate at 72°C for 25 min, then transfer the tube to ice.
Ligation of the A-tailed DNA Fragment to the T-vector
Usually 50-60 ng of T-vector is enough for each ligation. A
1:2 molar ratio of T-vector to insert DNA is recommended, but do not add more than 2 µl of A-tailed DNA solution in a 10 µl ligation (see notes in previous section).
1. In a sterile 0.5 ml microcentrifuge tube, add 10 X ligation buffer 1 µl A-tailed DNA (from previous step) ²2 µl T-vector 60 ng T4 DNA ligase (2-3 U/µl) 1 µl Sterile water to final total volume of 10 µl
Mix gently and centrifuge briefly.
2. Incubate at 14°C for 16 h or overnight. You may store the tube at -20°C if you do not plan to transform the cells immediately.
Transformation of the Ligated Vector
LB agar plates with appropriate antibiotics (most commonly 100 µg/ml of ampicillin) should be available and warmed to room temperature. If a plasmid with blue/white color selection is used, the LB plates should be spread with 5-bromo-4-chloro-3-indolyl-_-D-galactoside (X-Gal) and isopropylthio-_-D-galactoside (IPTG) before transformation. A 42°C water bath should be available for the transformation.
When a control ligation reaction with the T-vector alone is transformed, a considerable number of colonies may be obtained. When ligating inserts to the T-vectors, up to 90 % colonies should be positive. A successful ligation and transformation should yield hundreds of colonies. When few colonies are obtained, the ligase activity or the competency of the cells should be checked.
1. Thaw on ice one 50 µl aliquot of appropriate frozen competent cells (for example, Top 10F') for each
2. Add 2 µl of 0.5 M _-mercaptoethanol to the competent cells and mix by stirring gently with the pipette tip.
3. Add 2 µl of the ligation reaction to the competent cells, and mix gently by stirring with the pipette tip.
4. Incubate on ice for 30 min.
5. Heat shock the cells for 30 s in the 42°C water bath, then immediately place the cells on ice for 2 min.
6. Add 450 µl of SOC medium to the transformed cell vial.
7. Shake the cells at 37°C for 45-60 min at 225 rpm in a rotary shaking incubator.
8. Spread 100 µl of the transformed cells on each labeled LB-ampicillin plate. If plasmid with blue/white selection is used, the LB-plates should be spread with X-Gal and IPTG before spreading the cells.
9. Invert the plates and place them in a 37°C incubator overnight.
Identification of Positive Colonies
After transformation, the positive colonies may be identified either by restriction mapping of the miniprepared plasmids or screening by PCR. PCR screening can be carried out with primers that flank the vector cloning sites. In the case of the pBluescript plasmid, T3 and T7 primers may be used (19). The length of the amplified PCR product of a positive clone will be the size of the insert plus the bases flanking the two primers on the plasmid. This method is rapid, but may not work in some cases, especially when the insert is very large. Specific primers for the cloned insert may also be used as PCR screening primers. For screening by restriction mapping, plasmids are miniprepared, the insert is released by digestion with two unique restriction enzymes from the multiple cloning sites, and the insert size is confirmed by agarose gel electrophoresis. The orientation of the insert in the plasmid can also be determined by appropriate restriction digestion, in some cases. If needed, the orientation and identity of the clone may be further confirmed by sequencing. Because of the high cloning efficiency, in most cases only four to six colonies need to be screened.
Screening by Restriction Mapping
1. Randomly pick 4-6 colonies (in case of blue/white color selection, pick white colonies), and grow overnight at 37°C in 4 ml of LB medium with 50 µg/ml of ampicillin.
2. Pellet 2-3 ml of the cells by centrifuging for 2 min at top speed in a microcentrifuge. Isolate plasmids using Wizard Minipreps DNA purification system. The remaining cells may be stored at 4°C for 1-2 days. For long term storage, add glycerol to 15%, and store at -70°C.
3. Isolate plasmid DNA and digest 10-50% of the total preparation with the appropriate restriction enzymes.
4. Run an agarose gel to determine which colonies have the correct insert size and/or orientation.
5. Confirm the clone by sequencing when necessary.
S. 1991. Construction of T- vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1154.
12. Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A. and Smith, L.M. 1991. A universal method for the direct cloning of PCR amplified nucleic acid. Bio/Technology.
13. Clark, J. M. 1988. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16: 9677- 9686.
14. Hu, G. 1993. DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 12: 763-770.
15. Zhou, M.Y., Clark, S.E. and Gomez-Sanchez, C.E. 1995. Universal cloning method by TA strategy. BioTechniques. 19:34-35.
16. Hadjeb, N. and Berkowitz, G.A. 1996. Preparation of T-overhang vectors with high PCR product cloning efficiency. BioTechniques. 20: 20-22.
17. Erlich, H.A. 1989. PCR Technology. Stockton Press, New York, NY.
18. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White,
T.J. 1990. PCR Protocols: A guide to Methods and Applications. Academic Press, Inc., San Diego, CA.
19. Trower, M.K. and Elgar, G.S. 1994. PCR cloning using T-vectors. In: Methods in Molecular Biology, Vol.31: Protocols for Gene Analysis. A.J. Harwood, ed. Humana Press Inc., Totowa, NJ.
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