Preparation experiments for cloning vectors
Preparation experiments for cloning vectors
Many vectors and their derivatives have been developed for cloning PCR products, including the typical pBluescript-like vectors. These include the typical pBluescript vectors, which have multiple cloning sites and simplified multiple cloning sites, as does the PCR-Script Direct plasmid. Simplified polycloning sites allow the user to incorporate commonly used restriction enzyme sites into PCR primers, while also avoiding the problem of the same target sequence appearing in the plasmid vector at the same time. This experiment was derived from PCR Lab Guide (Second Edition) by Seed Kang and Lijia Qu.
Operation method
Preparation experiments for cloning vectors
Materials and Instruments
Chloroform-isoamyl alcohol Phenol: Chloroform-isoamyl alcohol (25:24:1) Lithium chloride (LiCl 10mol L) Phenol (Tris-saturated) TE buffer Ethanol Alkaline phosphatase Flat end restriction endonuclease and reaction buffer Cloning vector Move I. Materials For more product details, please visit Aladdin Scientific website.
Water baths Vacuum dryers Agarose gel electrophoresis equipment and reagents
1. Buffers, solutions and reagents
Chloroform-isoamyl alcohol (24:1)
Phenol: chloroform-isoamyl alcohol (25:24:1)
Lithium chloride (LiCl,10mol/L)
Phenol (Tris-saturated), pH 8.0
TE buffer (5 mmol/L Tris-HCl, pH 8.0, 0.1 mmol/LDTA)
Ethanol, 100%
2. Enzyme and enzyme buffer
Alkaline phosphatase (0.1~0.2U) (for Method B)
Commercially available molecular biology grade alkaline phosphatase usually contains nuclease contamination. Bacterial alkaline phosphatase is recommended, as it has been purified to remove nuclease contamination and is important for quality control in PCR-Script assays.
Flat-end restriction endonuclease (10-20U) and reaction buffer, i.e., Srf I or Sma I Universal Buffer, 10X (for Method B, which needs to be consistent with the restriction enzyme and alkaline phosphatase used) (1 mol/L potassium acetate, 250 mmol/L Tris-acetate, pH 7.6, 100 mmol/L magnesium acetate, 5 mmol/L/β-mercaptoethanol, 100ug/ml BSA). ,100ug/mlBSA)
3. Nucleic acids and oligonucleotides
Cloning vector (1ug)
4. Specialized equipment
3 water baths, one set to the appropriate temperature before the restriction enzyme and alkaline phosphatase treatments (Method B) and the other set to 65°C.
Vacuum dryer
5. Additional items
Optional: agarose gel electrophoresis equipment and reagents including ethidium bromide
II. METHODS A-Preparation of bidirectional cloning vectors .
The flat-end cloning procedure applies a cloning vector containing flat ends to capture DNA fragments for bidirectional insertion. Thus, the flat-ended vector does not require the nucleotide protrusions used to clone the inserted fragments. Flat-ended DNA fragments, such as PCR products, can then be cloned directly into these vectors. Flat-end cloning is inherently a very inefficient method, with recombinant inserts typically accounting for less than 10% of the overall transformants [see Figure 28-2(a)]. Efficiency can be improved by adding a restriction enzyme to the ligation system, as in the PCR-Script method [see Figure 28-2(b)] (Liuand Schwartz1992). 
1. Digest 1 pg of vector DNA with the appropriate flat-ended restriction endonuclease in the following 20ul reaction mixture.
Vector DNA (lug/ul) 1ul
Restriction enzyme (10U/ul) 1ul
Reaction buffer, 10X 2ul
H20 replenished to 2ul
2. Digest in a warm bath at the appropriate temperature for 1h.
Optionally, 1ul of the digested product can be used for agarose gel electrophoresis to detect the degree of linearization of the carrier DNA.
3. Extract the digestion product with phenol/chloroform, then add an equal volume of Tris saturated phenol (pH 8.0), shake vigorously, and transfer the upper liquid phase to a new tube. An equal volume of chloroform was added to the tube and shaken. Centrifuge briefly and carefully transfer the upper liquid phase to a new tube.
4. Heat treat the extracted DNA at 65°C for 20 min, removing any residual chloroform (boiling point of chloroform is 55°C).
5. Add 0.1 times the volume of 10mol/L LiCl and 2.5 times the volume of ice-cooled 100% ethanol to the DNA extract from step 2 to precipitate the DNA. mix gently and centrifuge at 12000 g for 10 min at room temperature.
6. After centrifugation, carefully pour out the supernatant. Drain the DNA precipitate for 10 min under vacuum.
7. Resolve the DNA with 5ul of TE buffer, and store the pre-digested bi-directional cloning vector at -20°C.
The final concentration of the digested DNA should be approximately 10ng/ul when the DNA is reconstituted with 50ul of TE buffer.
III. Method B ------- Directed Cloning Vector
A directed cloning method has been developed based on the known properties of T4DNA ligase and E. coli transformation, which does not require the addition of extra bases to the primers. First, T4DNA ligase requires both a phosphate group at the 5' end and a hydroxyl group at the 3' end to efficiently join the two strands of DNA. Second, the efficiency of linear DNA transformation of rcBC-deficient E. coli hosts is very low (approximately 4 orders of magnitude lower). Therefore, it is theoretically possible to achieve targeted cloning by preparing a single phosphorylated vector and a single phosphorylated insert fragment. Ligation of the insert into the vector in the desired direction produces a monocleaved circular molecule; if inserted in the undesired reverse direction, the ligation reaction produces a linear molecule that transforms E. coli very inefficiently. Treatment of the vector with one restriction endonuclease, removal of the exposed 5' phosphate with alkaline phosphatase, and subsequent digestion of the vector with another restriction endonuclease produces a monophosphorylated vector. Simplex restriction endonucleases can also be used in this way.
Proper DNA sequence manipulation allows enzyme-treated vectors to be used in PCR-Script-like reactions because self-conjugated vectors are easily digested by endonucleases in the conjugation reaction system and because the reading frame of the reporter gene is conserved. Because vector self-conjugation creates a restriction enzyme site, the need to use a highly purified enzyme for targeted and bidirectional cloning operations should not be overestimated. Nuclease contamination must be detected and removed prior to performing these experiments. As a particular example, the PCR-Script Direct targeted cloning method was applied to an SK(+) polyclonal site that was digested so that it contained both a Srf Ⅰ (5'-GCCC|GGGC-3') site and a SmaI (5'-TCCC 丨 GGGC-3', underlining indicates the Sam Ⅰ target sequence) site (Weiner 1993). Weiner 1993). The vector was first digested with Srf Ⅰ, followed by an alkaline phosphatase to remove the phosphate and a second digestion with SmaI. The short DNA fragments resulting from the Srf Ⅰ-Sam Ⅰ digestion are removed, and the Srf Ⅰ site is retained (see Figure 28-3). Because the reading frame is conserved, phenotypic selection can still be used. Monophosphorylated vectors can be prepared according to the following general guidelines.
1. Digest the appropriate vector DNA with the first flat-end restriction endonuclease (Srf I) in the following system.
Vector DNA (1ug/ul) 1ul (sic: 1ug/ul. - Editor's change)
Restriction enzyme (10U/ul) 1~2ul
Universal buffer, l0X 2ul
H20 replenished to 50ul
2. Warm bath at appropriate temperature for 1h.
Optionally, 1ul of the digestion product should be subjected to agarose gel electrophoresis to detect the degree of linearization of the vector DNA.
~undefined Optimize enzymatic processing of vector DNA using a buffer that can be used for both the first restriction endonuclease digestion and the alkaline phospholipase dephosphorylation reaction.
3. Inactivate the restriction enzyme in a warm bath at 65°C for 20 min and place on ice.
4. Add alkaline phosphatase (0.1~0.2U) directly to the heat-treated mixture and then warm bath as described in the procedure guide. 
5. extract the plasmid DNA that has been digested with restriction enzymes and treated with alkaline phosphatase with phenol/chloroform. then add an equal volume of Tris saturated phenol, shake vigorously, and transfer the upper liquid phase into a new tube. Add an equal volume of chloroform to the tube and shake. Heat treat the extracted DNA at 65°C for 20 min, removing any residual chloroform.
6. Set up a second 30-ul restriction enzyme digestion system containing the vector DNA that has been treated with alkaline phosphatase and the downstream flat-end restriction endonuclease, i.e., add 15 ul of DNA that has been extracted with phenol/chloroform, H20, 1X general purpose buffer, and the enzyme (10 to 20 U). Digest in a warm bath for lh at the recommended temperature.
Inactivate the second restriction enzyme in a warm bath at 7.65°C for 20 min and place on ice.
8. precipitate monophosphorylated DNA by adding 0.1x volume of 10mol/LLiCl and 2.5x volume of 100% ethanol pre-cooled on ice. mix gently and centrifuge at 12000 g for 10 min at room temperature.
9. After centrifugation, carefully pour out the supernatant. Evacuate the DNA precipitate under vacuum for 10 min.
10. Solubilize the DNA with 25ul of TE buffer.
The final concentration of monophosphorylated DNA should be approximately 10 ng/ul when the DNA is solubilized with 25ul of TE buffer. Store the monophosphorylated Targeted Cloning Vector at -20°C.