Preparation of insertion fragment experiments
Preparation of insertion fragment experiments
This experiment describes the preparation of insertion fragments. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Preparation of insertion fragment experiments
Materials and Instruments
Buffers, solutions and reagents Enzymes and enzyme buffers Nucleic acids and oligonucleotides Move Phase 1: Kinase processing of DNA primers For more product details, please visit Aladdin Scientific website.
Specialty equipment
It has been shown that many DNA polymerases (e.g., T7, modified T7, Taq, Vent, Tth, and Klenow) have deoxynucleotide terminal transferase (Tdt) activity (Clark 1988; Hu 1993). DNA polymerases extend nucleotides at the 3' end of a PCR product, a reaction that is nucleotide- and polymerase-specific. The reaction is nucleotide- and polymerase-specific. For example, Taq DNA polymerase produces a PCR product that is more likely to be modified in the following form (+ for extension; 1 for no nucleotide addition). 
There may not be a consistent pattern of bases added to the template by the polymerase. Therefore, it cannot be assumed that all DNA polymerases can be used to produce flat-ended DNA fragments. However, for some DNA polymerases, it is possible to control which nucleotide is obtained at the 3' end of the PCR product by using the nucleotide at the 5' end of the PCR primer (Hu1993; Costa and Weiner1994d). For targeted cloning using monophosphorylated vectors, monophosphorylation of the insert fragment can be achieved by treating the primers with kinase prior to PCR. Even better, a 5' phosphate group is added chemically during the synthesis of the PCR primers. Synthesizing PCR primers with a 5'-phosphate group ensures that all single-stranded DNA is monophosphorylated. The advantage of the kinase treatment is that all existing sets of primers can be modified, allowing for the application of targeted cloning of monophosphorylated vectors. the T4 polynucleotide kinase treatment is a simple and rapid technique.
I. MATERIALS
1. Buffers, solutions and reagents
ATP (10 mmol/L)
2. enzymes and enzyme buffers
Kinase buffer (lmmol/L magnesium chloride, 100 mmol/L Tris-HCl, pH 7.5, 5 mmol/L dithiothreitol)
T4 polyribonucleotide kinase (10U)
3. Nucleic acids and oligonucleotides
Oligonucleotide primers (1ug/ul)
4. Specialized equipment
Two water baths, pre-set to 37°C and 95°C
II. Methods
1. Add the following ingredients to the centrifuge tube.
Kinase buffer, 10X 3ul
ATP, 10mmol 0.5ul
T4DNA kinase (10U/ul) 1ul
Primer of choice 5ug
H20 supplemented to 30ul
2. 1h at 37°C.
3. Boil the reaction system at 95°C to inactivate T4DNA kinase.
Phase 2: PCR amplification
Since there are no operating guidelines that indicate which buffer conditions can be used for different types of DNA primer-template systems, a range of PCR buffers are usually tested. A number of PCR optimization kits are available for testing several different buffer components [e.g., Opti-Prime PCR Optimizer (Stratagene) and The PCR Optimizer (Invitrogen)]. The yield and specificity of PCR products can be improved by varying some of the PCR buffer components and utilizing PCR hot-start enzymes. In addition, the addition of betaine at a final concentration of 0.8-1.6 mol/L improves the efficiency of DNA amplification because betaine inhibits the formation of secondary structures in GC-rich regions.
I. Materials
1. Buffers, solutions and reagents
Betaine solution, PCR grade (Sigma)
dNTP stock solution (contains all 4 dNTPs, 100 mml/L each)
TE buffer (10 mmol/L Tris-HCl, pH 7.5, lmmol/LTTA, pH 8.0)
2. Enzyme and enzyme buffer
P/mDNA polymerase for cloning (Stratagene)
PCR buffer, 10X (purchased with the enzyme) or PCR optimization buffer; e.g., Opti-Prime PCR Optimization Kit (Stratagene) or The PCR Optimizer (Invitrogen)
Heat-stable DNA polymerase (5U); e.g., Taq DNA polymerase, TaqBead Hot Start Polymerase (Promega), High-Poly Platinum Taq DNA polymerase (Invitrogen), AmpliTaq Gold (Applied Biosystems)
3. Nucleic acids and oligonucleotides
Oligonucleotide primers
Template DNA (1~50ng plasmid)
4. Specialized equipment
PCR instrument
5. Additional items
Agarose gel electrophoresis equipment and reagents, including ethidium bromide
Optional: PCR enhancers, e.g., Taq Extender PCR additive (Stratagene), PCRx Enhancement System (Invitrogen)
II. Methods
1. On ice, set up a 25ul system in a sterile 0.5 ml (ul) centrifuge tube. Add the following reagents in order.
10X DNA polymerase buffer (eventually 1X) 2.5ul
Template DNA (1~50ng plasmid or 105~106 target molecule) 1~10ul
dNTP stock solution (contains all 4 dNTPs, 10 mmol/L each) 0.5ul
Betaine solution (5 mol/L) 2.5ul
Upstream primer (4ug/ul) 0.25ul
Downstream primer (4ug/ul) 0.25ul
Heat-stabilized DNA polymerase (5U/ul) 0.25ul
Optional: Taq Extender PCR Additive (5U) (extra) 0.25ul
H20 added to 25ul
~undefined For 3X105 targets: 1ug human single-copy genomic DNA; 10ng yeast DNA; 1% M13 phage.
2. Mix well and PCR amplification should be performed immediately.
3. Perform PCR on a PCR instrument with a thermal cover (if no thermal cover is available, the reaction system should be covered with mineral oil) and set the temperature program as recommended below. 
4. After the temperature cycle is complete, the PCR products are tested for fidelity and yield by agarose gel electrophoresis. 1-5 ul of PCR product contains DNA fragments that can be detected by staining with ethidium bromide in agarose gel electrophoresis. Control DNA of known concentration can be used as a reference for the size and concentration of the PCR product.
Stage 3: Polishing the ends of the PCR product with Pfu DNA polymerase
Optimization of primers for certain DNA polymerases can only minimally increase the number of flat-end fragments in PCR. The use of conventional Klenow polymerase for end polishing should be absolutely avoided, as it retains a fairly high level of exonuclease activity. Fortunately, T4 and PfuDNA polymerases do not have any DNA exonuclease activity or Tdt activity, so they can be used to produce flat ends after PCR (see Fig. 28-4) (Hu1993; Costa and Weiner1994c,d). 
PCR polishing involves removing the 3' end nucleotide extension of all PCR products with DNA polymerase. The resulting polished molecules can be ligated to flat-end cloning vectors with high efficiency by T4DNA ligase. Polishing the ends of PCR products prior to ligation increases the efficiency of the overall recombinant clone (Costa and Weiner 1994c,d; Weiner et al. 1994).
Pfu DNA polymerase is essentially inactive below 50°C. This allows the ligation reaction to be carried out at 4°C. This allows the ligation reaction to take place at 4 to 25°C and to proceed directly to the ligation reaction after the 72°C Pfu polishing step. This eliminates the need to extract the enzyme prior to the ligation reaction, which is required when polishing with T4 DNA polymerase. With a small amount of PCR product, Pfu polishing can produce high-fidelity, flat-ended DNA fragments in as little as 30 min. The general procedure for Pfu polishing is summarized below and can be performed either directly after PCR or after purification of the PCR product.
Prior to Pfu polishing, it is advisable to perform agarose gel electrophoresis to estimate the approximate concentration of the PCR product and to determine its correctness.PCR polishing uses only a fraction of the PCR amplification reaction product and is capable of polishing all DNA fragments present in the system. In a typical 25-ul PCR amplification reaction, 5 to 10 ul of product is used for polishing.
Since conventional PCR cloning procedures require a small amount of DNA insert (1-4ul), 5-10M1 of the PCR product can be used directly in an end-polishing reaction. If the end-polishing reaction is performed directly after PCR, there is enough dNTP and reaction buffer left over from the temperature cycle to perform the polishing reaction (see below). Precipitation or gel recovery of the PCR product can also improve the efficiency of end-polishing; however, if the purified PCR product is used for end-polishing, a mixture of reaction buffer and dNTP is required.
I. Materials
1. Buffers, solutions and reagents
dNTP stock solution (contains all four dNTPs at 100 mmol/L each), Method B
2. enzymes and enzyme buffers
Clonated Pfu DNA polymerase (2.5 U/ul)
Clonized Pfu buffer, 10X (company supplied)
3. Nucleic acids and oligonucleotides
PCR products, purified (Method B) and unpurified (Method A)
4. specialized equipment
Water bath, pre-regulated to 72°C
ii. Method A-Polishing of unpurified PCR products
1. To polish PCR-produced DNA fragments, transfer the PCR products directly from the PCR reaction tube to a 0.5 ml sterile centrifuge tube and add the following reagents in the order listed below.
5-10ul of PCR product
1ul cloned Pfu DNA polymerase (2.5U)
Add ddH20 to a final volume of 10ul
Gently mix the components and warm bath with a hot lid (if no hot lid is available, the reaction system should be covered with mineral oil)
2.72°C warm bath for 30 min, light polishing reaction.
3. After 30 min warm bath, place the reaction on ice.
4. The end-polished DNA fragments can be added directly to a ligation reaction.
Method B-Pfu polishing of purified PCR products
1. To polish the purified PCR product, the precipitated PCR product is pipetted into a sterile 0.5 ml centrifuge tube and the following reagents are added sequentially.
5 to 10 ul Precipitated PCR product
1ul 10X Cloning Pfu DNA Polymerase Buffer
1ul dNTP mixture (10 mmol/L overall, 2.5 mmol/L per nucleotide triphosphate)
1ul cloned DNA polymerase (2.5U)
Replenish with ddH20 to a final volume of 10ul
Gently mix the components and warm bath in a hot lid (if not warm bathed in a hot lid, you need to cover the reaction with mineral oil)
2.72°C bath the reaction system for 30 min.
3. After the 30 min bath, place the reaction system on ice.
4. End-polished DNA fragments can be added directly to the ligation system.
PCR product purification (optional)
The percentage of recombinants can be increased by removing excess PCR primers by ammonium acetate precipitation prior to the cloning protocol. The PCR product can be salted out as described below.
I. Materials
1. Buffers, solutions and reagents
Ammonium acetate, 4 mol/L
Ethanol, 100% and 70
STE buffer, 10X (1 mol/L NaCl, 200 mmol/L Tris-HCl, pH 7.5, 100 mmol/L EDTA)
TE (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, pH 8.0)
2. Specialized equipment
Vacuum dryer
II. METHODS
1. Add 0.1 times the volume of STE buffer.
2. Add an equal volume of 4mol/L ammonium acetate to the sample.
3. Add 2.5 times the volume of 100% ethanol equilibrated at room temperature.
4. Immediately precipitate the DNA by centrifugation at 12000 g for 10 min at room temperature and carefully pour off the supernatant.
5. Add 200ul of 70% (v/v) ethanol.
6. Centrifuge at 12000 g for 10 min at room temperature, pour off the supernatant carefully and dry the precipitate in its vacutainer.
7. Resuspend the DNA in the original volume of TE buffer and store at 4°C.