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Cloning 1 (Ligation)

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Cloning 1 (Ligation) Cloning 1 (Ligation) Objectives: a) to learn the strategies of cloning b) to clone the 1.3 kb fragment from the TTG gene into a cloning vector What is cloning? According to Paul Berg (Nobel Laureate): “We use the word cloning in science as a term to describe the production of many copies of a starting material”. What is a clone? Clone is a group of cells that stem from a single cell. Clone: identical in genetic make up What is a clone? Clone is a group of cells that stem from a single cell. This is not a clone! In order to clone a gene: 1. Isolate the DNA of interest. 2. Introduce the DNA into a cloning vector (such as a plasmid), thus create a recombinant plasmid. A plasmid is a double-stranded DNA that can gain admission to, and replicate in, the cytoplasm of many kinds of bacterial cells. A cloning vector is a modified plasmid, capable of replication in a host organism (such as bacterium), and into which a piece of foreign DNA can be inserted. Z Y Polylinker X U E H B P S lacZ Ori Ori Plasmid Vector Antibiotic Antibiotic resistance resistance gene gene 2. Introduce the DNA into a cloning vector to create a recombinant vector. E E E E E Vector DNA Cut with enzyme E Cut with enzyme E Vector Mix Vector Vector Vector Vector Recombinant vectors Non-recombinant vector 3. Introduce recombinant/non-recombinant vectors into E. coli competent cells (transformation). E. coli competent cell Vector Vector Vector Vector 4. Propagate transformed E. coli cells in SOB (or SOC) medium. E. coli containing recombinant vectors E. coli containing non-recombinant vectors E. coli without vectors 5. Culture propagated E. coli cells on agar plates containing antibiotic and Xgal (color indicator). Let grow overnight at 37°C. Agar plate containing antibiotic Blue colony: E. coli cells containing non-recombinant vectors White colony: E. coli cells containing recombinant vectors No colony: E. coli cells without vector (i.e., antibiotic sensitive) die in antibiotic-rich medium In today’s experiment you will be using a new type of vector called TOPO®. TOPO has two advantages over the pUC19 vectors: a) it is linear b) it has single, overhanging 3’ deoxythymidine (T) residue PCR product G A A A A G T T T T C C TOPO Since the Taq polymerase, used in the PCR reaction, adds a single deoxyadenosine (A) to the 3’ ends of the PCR product, this allows the product to ligate efficiently with the vector (A T ligation). Procedure 1. Use 6 μLof the PCR product (prepared in Lab 5) plus 2 μL of gel loading dye for electrophoresis; load the gel as shown below: DNA PCR product ladder 1 2 3 Gel DNA PCR product ladder 1 2 3 2. Electrophorese the gel for 40 min at 90 volts. 3. Photograph the gel, view the bands, and save the tubes that show the 1.3 kb fragment. The gel must be consistent with the figure shown below. TOPO vector, 1 μL Cloning dH O, 1 μL 2 reaction PCR product, 3 μL Ligation buffer, 1 μL -mix gently -centrifuge briefly -room temperature 5 min -ice 2 min Meanwhile, place E. coli competent cells on ice to thaw: it takes about 5 min. 2 μL Tube containing 100 µL of competent cells Cloning reaction -mix gently, do not pipette -ice 10 min -42°C water bath 45 sec -ice 900 μL Add SOB medium Competent cells + cloning reaction 37°C shaker for 40 min at 225 rpm Transfer 50 μL to an agar plate (LB + kan + Xgal) 50 μL Competent cells + cloning reaction + SOB medium -spread well -incubate at 37°C overnight -check plate for white & blue colonies Why Blue and White Colonies? The wild-type strain of E. coli has the gene lacZ which codes for the enzyme ß-galactosidase (ß-gal). lacZ E. coli To indicate the expression of ß-gal, an organic compound is used which is called X-gal (5-bromo-4-chloro-3-indolyl-beta-D- galactopyranoside “BCIG”). ß-gal X-gal 5-bromo-4-chloro-3-hydroxyindole galactose dimerized 5, 5’-dibromo-4, 4’-dichloro-indigo (blue color) How does the lacZ gene work in TOPO vector? LacZ gene codes for ß-gal -codes for the -codes for the amino group of ß-gal carboxyl group of ß-gal Polylinker mutant E. coli TOPO -does not code for a -does not code for a functional ß-gal functional ß-gal 1. Transform E. coli with non-recombinant TOPO vector (the vector does not carry an insert DNA) Polylinker without insert E. coli chromosome TOPO E. coli in medium containing X-gal -functional ß-gal -blue color 2. Transform E. coli with recombinant TOPO vector (the vector does carry an insert DNA) Insert TOPO E. coli chromosome E. coli in medium containing X-gal The insert interrupts the reading frame of the gene; therefore: -defective ß-gal -no blue color (white) E. coli cells with non-recombinant vectors E. coli cells with recombinant vectors Agar plate containing X-gal.
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