LAB 9 RECOMBINANT DNA LIGATION

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1 BIOECHNOLOGY I RECOMBINN DN LIGION LB 9 RECOMBINN DN LIGION SUDEN GUIDE GOL he objective of this lab is to perform DN ligation to construct a recombinant plasmid. OBJECIVES fter completion, the student should be able to: 1. Perform DN ligation. 2. Explain the steps of ligation of a recombinant plasmid. 3. Explain why plasmid vectors are sometimes 5 -dephosphorylated before ligation. 4. List and describe the important structures found on plasmid vectors used in many biotech laboratories. BCKGROUND he cloning of a gene first involves restriction enzyme digestion of chromosomal DN. he restriction fragment containing the gene of interest is isolated from the remaining genomic DN. Fragments of the correct size are then joined, or ligated, with larger fragments of DN, called vectors, creating recombinant DN. he vector DN can be phage (bacteria-infecting virus), a plasmid, or a combination of the two (cosmids and phasmids). Bacterial plasmids are small naturally occurring circular DN molecules capable of replication independent of the bacterial chromosome. Plasmids frequently carry genes that benefit the bacterium, such as those bestowing antibiotic resistance. Once the fragments are ligated into the plasmid, bacterial cells are transformed by manipulations designed to take the recombinant DN across cell membranes and into the cytoplasm. However, many cells will not be transformed and those that are transformed may contain plasmids without the gene of interest. Finding cells that have a replicated plasmid that contains the gene of interest would be like looking for a needle in a haystack, if it were not for selection techniques. Because plasmid vectors carry a gene for antibiotic resistance, cells with a plasmid can be selected by using an antibiotic to kill any cells that were not transformed. Screening for cells with insert DN is accomplished by Southern hybridization, assaying for the product of the recombinant gene or by bluewhite selection. Eilene Lyons Revised 1/12/2010 Page 9-1

2 BIOECHNOLOGY I RECOMBINN DN LIGION LIGION Ligase is an enzyme derived from a phage. It requires P in a biochemical reaction that joins two fragments of DN together by forming a phosphodiester bond between the 5 phosphate and the 3 hydroxyl groups of adjacent nucleotides. he formation of a phosphodiester bond is an endergonic reaction, i.e., it requires energy, which is why P must be added. Ligase, like many enzymes, requires a magnesium ion cofactor for optimal function. Cloning requires the joining, or ligation, of fragments with complementary ends or blunt ends generated by restriction enzyme digestion. When different DN fragments are digested with the same restriction enzyme that yields sticky ends, the ends are compatible and therefore will ligate. Sticky ended fragments resulting from the digestion of two different enzymes can be ligated only if the ends are compatible (see Figure 1). CC GG Enzyme X site CC GG a. b. CC GG a. d. = CC GG CC GG Enzyme Y site. Compatible Sticky Ends CC GG c. d. Results of digestion CC GG c. b. = CC GG Results of ligation GGC CCG Enzyme Z site GGC CCG a. b. CC CCG a. d. CC CCG CC GG Enzyme Y site B. Incompatible Sticky Ends CC GG c. d. Results of digestion GGC GG c. b. No ligation GGC GG Figure 1. Compatible and Incompatible Sticky End Ligation Eilene Lyons Revised 1/12/2010 Page 9-2

3 BIOECHNOLOGY I RECOMBINN DN LIGION Enzymes that yield blunt ends give fragments that will ligate with any other blunt ended fragment. When ligating an insert fragment of DN into a plasmid vector, the molar ratio of insert DN should be 3-4 times that of the plasmid vector. his helps to ensure there is sufficient insert DN available to produce recombinant molecules and decreases the likelihood of self-ligation of the plasmid vector when its ends are compatible. Dephosphorylating with alkaline phosphatase can also prevent production of self-ligated single and concatamer plasmid vector. ( concatamer is two or more linear plasmids ligated together. Insert DN can be included in this wild bit of ligation, which can form huge rings.) Dephosphorylation of the linear plasmid removes the 5 phosphates, which are required by the ligase enzyme in order to catalyze the formation of a phosphodiester bond between nucleotides. Insert DN will still ligate into the vector because the insert has its own 5 phosphates, so there is joining to the vector plasmid at each end of the insert DN. he 3 end of each insert DN strand will not ligate to the vector s 5 end because the 5 phosphate is missing. nick, which is a break along one side of the DN double helix, will be present on each strand of the recombinant DN plasmid. See Figure 2. Enzymes in the cell repair these nicks once the plasmid has been transformed. Double Stranded Insert DN Nick where vector s 5 phosphate was missing ' Nick where vector s 5 phosphate was missing. Ligase worked here at insert 5 ends Double Stranded Vector DN Figure 2. Results of the ligation of insert DN into a vector that has been dephosphorylated. Eilene Lyons Revised 1/12/2010 Page 9-3

4 BIOECHNOLOGY I RECOMBINN DN LIGION CLCULING HE MOUNS OF INSER ND VECOR DN he following formula is used to calculate the amount of insert to use in a ligation reaction: ng insert DN to use in a ligation = (ng of vector) (size of insert in kb) X (desired molar ratio of insert:vector) (size of vector in kb) For example, an insert of 2.8 kb has a concentration of 40 ng/ l and a 3.2 kb vector has a concentration of 100 ng/ l. he amount of vector DN used will be 100 ng. 4:1 molar ratio is required. herefore, 1.0 l of vector DN and 8.75 l of insert DN will be used: Given: 40 ng/ l of 2.8 kb insert DN 100 ng/ l of 3.2 kb vector DN 100 ng vector DN used = 100 ng/100 ng/ l = 1.0 l vector DN ng insert DN to use in the ligation = (100 ng) (2.8 kb) X 4/1 = 350 ng insert (3.2 kb) (350 ng)/ (40 ng/ l) = 8.75 l insert DN If the calculated amount of insert DN is more than is available, decrease the number proportionately (e.g., 175 ng insert and 50 ng vector). he total volume of a ligation reaction is kept small, approximately 10 l, since the amount of insert DN is sometimes limited and the DN concentrations must be kept high. In a ligation reaction of 10 l total volume, 0.5 l of 4 Ligase, 10X 4 ligation buffer (1X final concentration), and 0.5 l [20 mm] P is added along with the insert and vector DN. Based on the concentration of the insert DN and the concentration of the digested vector DN, calculate the volume of each DN that must be added to ensure sufficient total DN and the correct insert: vector ratio. In the example given, 1.2 l 10X 4 ligation buffer, and no water, would give a total volume of l: Sample Ligation Reaction Setup 8.75 l insert DN (350 ng; assuming an initial concentration of 40 ng/ l) 1.0 l plasmid vector DN restriction reaction (100 ng; assuming 100 ng/ l) 1.2 l 10X 4 ligation buffer (1X final concentration) 0.5 l [20 mm] P 0.5 l 4 ligase l ddh 2 O, (may be required if DN is very concentrated) l total volume Eilene Lyons Revised 1/12/2010 Page 9-4

5 BIOECHNOLOGY I RECOMBINN DN LIGION LBORORY OVERVIEW In this lab, you will be cloning a fragment of DN from a phage genome into a puc plasmid. In the next lab, a transformation will be performed and blue-white selection will be used to determine if colonies contain recombinant plasmids. SFEY GUIDELINES GLP requires that you wear eye protection, gloves and a lab coat. MERILS per class 4 ligase Gene cleaned insert DN Digested plasmid DN Cooling block set at 16 C Vortex mixers one per each end of lab bench Scotch tape per group Molecular grade or qualified water 1.5 microcentrifuge tubes 10X Ligase buffer P 20 mm ice and ice bucket personal microcentrifuge 20 l micropipetter and sterile tips Sharpie marker 1.5 ml microcentrifuge tubes Microcentrifuge tube racks Scissors Eilene Lyons Revised 1/12/2010 Page 9-5

6 BIOECHNOLOGY I RECOMBINN DN LIGION PROCEDURE NOE: Each team will set up only one ligation reaction. eams 1 & 2 will also perform a control ligation reaction each. 1. haw your samples of digested plasmid DN and gene cleaned fragment DN. (NOE: If ligation takes place immediately after plasmid DN restriction, it must be heated at 65 C for 20 minutes to inactivate the restriction enzyme.) Centrifuge each briefly to collect the entire samples in the bottom of the tubes. 2. Label a 1.5-ml microcentrifuge tube as "ligation" and include the date, and your group number or initials. (he size of the tube depends on the incubator used in step 5, below. Check first on whether the thermal cycler or the cooling block is to be used.) 3. Completely thaw the 10 ligase buffer. DO NO USE BUFFERS H CONIN ICE PRICLES S HE BUFFER CONCENRION WILL NO BE CORREC. Vortex to mix and centrifuge briefly before opening the tube. Store on ice, along with the P. he 4 DN ligase enzyme must be kept in the -20 C ice block. 4. Use the guidelines in the background information to calculate the amounts of insert and plasmid vector to use in your ligation. Remember that the enzyme is in glycerol; pipette slowly and carefully. fter adding the enzyme, pipette the mixture up and down to mix. Centrifuge briefly. eam 1 should set up negative control ligation #1 using no insert (vector only). Use molecular grade water instead of the missing component. eam 2 should set up negative control ligation #2 using no ligase. dd molecular grade water instead of the missing component. hese will be the negative controls for the entire class. Gene cleaned fragment DN l Digested plasmid DN l 10 Ligase Buffer 1.0 l 20 mm P 0.5 l DN 4 Ligase 0.5 l otal volume 10 l 5. Incubate at 16 C for 1-2 hours in a cooling block. he optimum temperature of ligation is 16ºC but the reaction will occur if incubated at colder temperatures for longer periods of time. If you will not proceed to the next step after ligation immediately, the reaction can also be incubated at least 18 hours at -20ºC. Eilene Lyons Revised 1/12/2010 Page 9-6

7 BIOECHNOLOGY I RECOMBINN DN LIGION D NLYSIS here will be no data to analyze until the results of transformation are analyzed. Ligation will be classified as successful if recombinant plasmids are formed. his cannot be determined until transformants are grown in culture to clone the recombinant plasmids and the DN from those plasmids is purified and identified. QUESIONS 1. Explain why the ratio of insert to vector DN is so crucial to the success of genetic engineering of a recombinant plasmid. 2. Why must P be added in a ligation reaction? 3. Explain why plasmid vectors are sometimes 5 -dephosphorylated before ligation. 4. If a plasmid vector is dephosphorylated, explain how a DN fragment can be ligated into it, since it does not have the 5 phosphates that the ligase must recognize to form the phosphodiester bond. 5. When must a digestion be heated to 65 C prior to the DN s use in a ligation? Explain. 6. You are setting up a ligation of a 1.3 kb DN fragment into a 3.2 kb plasmid. he concentration of the insert DN (the 1.3 kb fragment) is 30 ug/ul and that of the plasmid vector is 80 ng/ul. Do the calculations and explain how you will set up the ligation reaction. Eilene Lyons Revised 1/12/2010 Page 9-7

8 BIOECHNOLOGY I RECOMBINN DN LIGION Eilene Lyons Revised 1/12/2010 Page 9-8

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