分子构建转化鉴定(分子生物学实验)

DNA Molecule Recombination and Transformation Lin ChengyuBio 04 2010030007; Cooperator: Yuan Xiaowen1Introduction1.1Background InformationDNA Molecule Recombination refers to the process that the target DNA is put into thevector using biochemical methods. Then recombinant DNA is transferred into theorganism such as E.coli in order to get the product. Identification is needed to preventfalse positive clone.1.2Objectives(1)Learn the method of DNA purification by ethanol precipitation;(2)Learn the method of DNA subcloning;(3)Learn the CaCl2 method of E.coli competent cells preparation;(4)Learn the methods of transformation and clone selection;1.3Major Principles1.3.1Ethanol precipitationDNA molecule recombination needs purified DNA in order to get high successratio. In the experiment we use ethanol precipitation, because big DNAmolecule is insoluble in high ethanol concentration solution. And smallfragment of DNA can be eluted using this property, while the target DNA willbe in the pellet.1.3.2LigationConnect the 5’ end and 3’ end from two different DNA fragments using DNAligase. The process is shown in Figure 1.Figure 1 DNA ligation1.3.3 E.coli competent cells preparationNaturally, bacterial cells are not easy to allow foreign DNA to pass their cellwalls. However, when they are treated with some reagents, such as CaCl2, theircell walls will be altered which makes the foreign DNA enter the cells easily.Such cells are called "competent cells”.1.3.4TransformationThe process of introducing foreign DNA into the competent cells is called“transformation”. In this experiment, heat shock method is used to introduce theforeign DNA into the competent cells prepared by CaCl2 through heat shock.2Experiment Operation2.1MaterialPCR productpBluescript KS plasmidNEB 1kb DNA ladderBam HI, Hin dIIITable 1 Recognition Sequence and Cutting Site of Bam HI and Hin dIII1E.coli DH5α2.2Chemicals and Apparatus2.2.1SolutionsTable 2 SolutionsTable 2 Solutions (Continued)2.2.2Apparatus(1)Pipettes;(2)Eppendorf tubes;(3)Electrophoresis apparatus(4)UV gel imaging system(5)Water bath2.3Procedure2.3.1Restriction enzyme digestionAdd all the components into the Eppendorf tube following Table 3Table 3 Scheme of restriction enzyme digestionIncubate the tubes at 37 ℃for 1 h.2.3.2Ethanol precipitation(1)Add 2-fold (40 μL) absolute ethanol and 0.1-fold (2 μL) 3 M to theEppendorf tube;(2)Incubate at -20 ºC for 10 min;(3)Centrifuge at 12,000 rpm for 10 min, 4 ºC;(4)Discard the supernatant carefully, add 100 μL ethanol, and resuspend thepellet;(5)Centrifuge at 12,000 rpm for 10 min, 4 ºC;(6)Discard the supernatant thoroughly, and leave the tube uncovered for 5min;(7)Add 10 μL ddH2O to solute the target DNA;(8)Run agarose gel electrophoresis using 2 μL target DNA and 1 μL vector.2.3.3Ligation(1)Decide the concentration of target DNA and vector by comparing the twobands;(2)Add all components to the Eppendorf tube following Table 4;Table 4 Scheme of ligation2.3.4 E.coli competent cells preparation(1)Add 1.8 mL culture medium containing E.coli. to the Eppendorf tube;(2)Centrifuge at 4,000 rpm for 1 min, 4 ºC;(3)Discard the supernatant, and add another 1.8 mL culture mediumcontaining E.coli;(4)Centrifuge at 4,000 rpm for 10 min, 4 ºC;(5)Discard the supernatant, add 1 mL ice-cold 0.1 M CaCl2 and resuspend;(6)Centrifuge at 4,000 rpm for 10 min, 4 ºC;(7)Repeat Step 5 and 6;(8)Discard the supernatant, and 200 μL ice-cold 0.1 CaCl2 and resuspend;(9)Incubate on ice;2.3.5Transformation(1)Add 100 μL solution containing competent cells to an Eppendorf tube;(2)Add 10 μL recombinant plasmid;(3)Mix gently and incubate on ice for 20 min;(4)Heat shock at 42 ºC for 90 sec;(5)Put the tube on ice immediately, and incubate for 2 min;(6)Add 900 μL LB culture without antibiotic;(7)Incubate at 37 ºC for 20 min;(8)Incubate the cells on a new plate containing ampicillin, X-gal, and IPTG; 2.3.6Identification2.3.6.1Plasmid extraction and purification(1)Select a positive clone (white in this experiment) and put into the LBculture medium containing ampicillin;(2)Incubate at 37 ºC overnight;(3)Add 1.2 mL bacterial culture into an Eppendorf tube, and centrifuge at10,000 rpm for 1 min;(4)Discard the supernatant;(5)Repeat step 3 and 4;(6)Add 100 μL Solution I, and resuspend the pellet using pipette andvigorously vortexing;(7)Add 200 μL of freshly prepared Solution II, and mix the contents byinverting the tube rapidly five times;(8)Incubate on ice for 5 min;(9)Add 150 μL ice-cold solution III, and mix the contents by inverting the tubeseveral times;(10)I ncubate on ice for 3~5 min;(11)C entrifuge at 10,000 rpm for 7 min.(12)T ransfer the supernatant to a new Eppendorf tube, and add 1 mL absoluteethanol;(13)C entrifuge at 12,000 rpm for 5 min, and discard the supernatant;(14)A dd 500 μL 70 ethanol, and resuspend using pipette;(15)C entrifuge at 12,000 rpm for 5 min, and discard the supernatant;(16)I nvert the tube on a paper towel to dry the tube at R.T.;(17)D issolve in 30~50 μL ddH2O or TE containing 20 μL/mg DNase-freeRNase A, and incubate at R.T. for 5 min.2.3.6.2Restriction enzyme digestion(1)Add each component following Table 5Table 5 Enzyme digestion of plasmid DNA(2)2.3.6.3Agarose gel electrophoresis(1) Prepare the agarose solution in TAE at the concentration of 0.8%;(2) Prepare a piece of agarose gel;(3) Add 10 μL loading buffer to 20 μL enzyme digestion product;(4) Load the sample as well as the DNA ladder to the slots;(5) Start electrophoresis at 100 V;3Result and Discussion3.1Ethanol precipitationThe gel image is shown in Figure 2Figure 2 Ethanol precipitationLane I: PCR productLane II: pBluescript KSLane M: NEB 1kb DNA ladderCompare Band I and II, they are about the same shade. Considering that the loadingsample of target DNA is 2 μL and vector 1 μL, the concentration ratio of target DNA andvector should be 1:2. During ligation, we add 1 μL vector and 2μL target DNA to maketheir molecule ratio in an appropriate level.3.2TransformationThe gel image is shown in Figure 3Figure 3 IdentificationLane I: Clone ILane II: Clone IILane M: NEB 1kb DNA ladderClone II contains a band at about 0.9 kb, which is expected as a positive clone, whichcan be used in the following process, while clone I is blank in the gel image. It is mainlyand possibly because some mistakes made in the plasmid extraction and purificationsteps.4ConclusionClone II has the recombinant DNA and is proper for the following process.5Reference【1】/。