重组DNA的分离、克隆与测序实验手册-5
C. Random fragment end-repair, size selection, and phosphorylation
Since both sonicated and nebulized DNA fragments usually contain single-stranded ends, the samples are end-repaired prior to ligation into blunt-ended vectors (10,11). A combination of T4 DNA polymerase and Klenow DNA polymerase are used to "fill-in" the DNA fragments by catalyzing the 3'-5' incorporation of complementary nucleotides into resultant double-stranded fragments with a 5' overhang. Additionally, the single-stranded 3'-5' exonuclease activity of T4 DNA polymerase is used to degrade 3' overhangs. The reactions included the two enzymes, buffer, and deoxynucleotides and are incubated at 37degC.
Following fragment end-repair, the DNA samples are electrophoresed on a preparative low-melting temperature agarose gel versus the phi-X 174 marker, and after appropriate separation, the fragments in the size range from 1-2Kbp and 2-4Kbp are excised and eluted separately from the gel, as discussed above. Alternatively, the fragments can be purified by fractionation on a Sephacryl S-500 spin column as also discussed above. In both instances, the purified fragments are concentrated by ethanol precipitation followed by resuspension in kinase buffer, and phosphorylation using T4 polynucleotide kinase and rATP. The polynucleotide kinase is removed by phenol extraction and the DNA fragments are concentrated by ethanol precipitation, dried, resuspended in buffer, and ligated into blunt-ended cloning vectors. It should be noted that because a significant portion of nebulized DNA fragments are easily cloned without end-repair or kinase treatment, these two steps can be combined without significantly affecting the overall number of resulting transformed clones (see section V.B. on purification of PCR fragments for cloning, which describes a method for simultaneous end-repair and kinase treatment).
Protocol
1. To each tube containing 35 ul of DNA fragments (five of sonicated DNA and four of nebulized DNA), add:
0.25 mM dNTPs 2 ul T4 DNA polymerase 3 ul (3 U/ul) Klenow DNA polymerase 2 ul (5 U/ul) 42 ul
T4 (203L) and Klenow (210L) DNA polymerases from New England Biolabs.
2. Incubate at room temperature for 30 minutes.
3a. Add 5 ul of agarose gel loading dye and apply to separate well of a 1% low gel temperature agarose gel and electrophorese for 30-60 minutes at 100-120 mA.
4a. Elute the DNA from each sample lane, ethanol precipitate, and resuspend the dried DNA in 36 ul of sterile ddH2O and add 4 ul of 10X denaturing buffer. There should be five tubes for sonicated fragments and four tubes for nebulized fragments.
5a. Incubate at 70degC for 10 minutes, and place the samples in an ice-water bath.
6a. Add the following reagents for the kinase reaction and incubate at 37 degC for 10-30 minutes:
10 mM rATP 1 ul 10 X kinase buffer 5 ul T4 polynucleotide kinase 1 ul (30 U/ul) Final Volume 47 ul
T4 polynucleotide kinase (70031) from United States Biochemicals.
7a. Pool the kinase reactions, phenol extract, ethanol precipitate, and resuspend the dried DNA fragments in 40 ul of 10:0.1 TE buffer. This yields a typical concentration of 500-1000 ng/ul.
Alternatively the end-repair and phosphorylation steps can be combined:
1b. Resuspend DNA in 27 ul of 1X TM buffer. Add the following:
10X kinase buffer 5 ul 10 mM rATP 5 ul 0.25 mM dNTPs 7 ul T4 polynucleotide kinase 1 ul (3 U/ul) Klenow DNA polymerase 2 ul (5 U/ul) T4 DNA polymerase 3 ul (3 U/ul) ------------------------------------------------------ Final Volume 50 ul note: if the DNA has been sheared by nebulizing, the T4 DNA polymerase addition here may not be necessary.
2b. Incubate at 37degC for 30 minutes
3b. Add 5 ul of agarose gel loading dye and apply to separate well of a 1% low melting temperature agarose gel and electrophorese for 30-60 minutes at 100-120 mA.
4b. Elute the DNA from each sample lane, ethanol precipitate, resuspend in 10 ul of 10:0.1 TE buffer.
D. DNA ligation
DNA ligations are performed by incubating DNA fragments with appropriately linearized cloning vector in the presence of buffer, rATP, and T4 DNA ligase (10,11). For random shotgun cloning, sonicated or nebulized fragments are ligated to either SmaI linearized, dephosphorylated double-stranded M13 replicative form or pUC vector by incubation at 4degC overnight. A practical range of concentrations is determined based on the amount of initial DNA, and several different ligations, each with an amount of insert DNA within that range, are used to determine the appropriate insert to vector ratio for the ligation reaction. In addition, several control ligations are performed to test the efficiency of the blunt-ending process, the ligation reaction, and the quality of the vector (10,11). These usually included parallel ligations in the absence of insert DNA to determine the background clones arising from self-ligation of inefficiently phosphatased vector. Parallel ligations also are performed with a known blunt-ended insert or insert library, typically an AluI digest of a cosmid, to insure that the blunt-ended ligation reaction would yield sufficient insert containing clones, independent of the repair process.
Protocol
1. Combine the following reagents in a microcentrifuge tube, and incubate overnight at 4degC:
DNA fragments 100-1000 ng cloning vector 2 ul (10 ng/ul) 10X ligation buffer 1 ul T4 DNA ligase (NEB 202L) 1 ul (400 U/ul) sterile ddH2O q.s. 10 ul
The cloning vector typically is SmaI-linearized, CIAP-dephosphorylated pUC vector (Pharmacia 27-4860-01) as several years ago we switched from M13 to pUC-based shotgun cloning. The advantage of obtaining two sequence reads off one isolated shotgun sub-clone seems to outweigh the disadvantage of a few bases less in double-stranded vs single-stranded read lengths. In some instances, including 5% PEG in the ligation reactions also seems to slightly improve the ligation efficiency.
2. Include control ligation reactions with no insert DNA and with a known blunt-ended insert (such as AluI digested cosmid).
E. Competent cell preparation
There are two main methods for preparation of competent bacterial cells (14) for transformation, the calcium chloride and the electroporation method. For the calcium chloride method, a glycerol cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media. This culture then is preincubated at 37degC for 1 hour, transferred to an incubator-shaker, and is incubated further for 2-3 hours. The cells are pelleted by centrifugation, resuspended in calcium chloride solution, and incubated in an ice-water bath. After another centrifugation step, the resulting cell pellet again is resuspended in calcium chloride to yield the final competent cell suspension. Competent cells are stored at 4degC, for up to several days.
Calcium Chloride Protocol
1. Thaw a frozen glycerol stock of the appropriate strain of E. coli, add it to an Erlenmeyer flask containing 50 ml of pre-warmed 2xTY (1) media, and pre-incubate in a 37degC water bath for 1 hour with no shaking. Further incubate for 2-3 hours at 37degC with shaking at 250 rpm.
2. Transfer 40 ml of the cells to a sterile 50 ml polypropylene centrifuge tube, and collect the cells by centrifugation at 3000 rpm for 8 minutes at 4deg C in a GPR centrifuge (Beckman) or 6000 rpm for 8 minutes at 4degC in an RC5-B centrifuge (DuPont) equipped with an SS-34 rotor. For M13-based transformation, save the remaining 10 ml of culture in an ice-water bath for later use.
3. After centrifugation, decant the supernatant and resuspend the cell pellet in one-half volume (20 ml) of cold, sterile 50 mM calcium chloride, incubate in an ice-water bath for 20 minutes, and centrifuge as before.
4. Decant the supernatant and gently resuspend the cell pellet in one-tenth volume (4 ml) of cold, sterile 50 mM calcium chloride to yield the final competent cell suspension.
Preparation of calcium chloride competent cells for frozen storage
1. Transfer 166 ul of the competent cell suspension to sterile Falcon culture tubes.
2. Add 34 ul of sterile 100% glycerol to the 166 ul aliquots of the final competent cell suspension prepared above, giving a final concentration of 17 % glycerol.
3. The competent cells then should be placed at -70degC and can be stored indefinately.
4. To use competent cells for transformation, remove from freezer and thaw for a few minutes at 37degC. Place on ice, add plasmid DNA and incubate for one hour as in the standard transformation procedure. Then heat shock at 42degC for 2 minutes, cool briefly, add 1 ml of 2xTY and incubate for 1 hour at 37degC before spreading on plates.
