Sub-cloning of Bacterial DNA to form Recombinant DNA
We performed a sub-cloning of DNA to create a recombinant DNA molecule. We isolated the DNA of choice, the insert, and place it into a vector, also known as a ligation. The plasmid and insert DNA was isolated using the alkaline lysis method of nucleic acid isolation. After that we isolated and purified the restriction digested vector and insert from a 1% agrarose gel by performing a gel electrophoresis. Next we performed a ligation, joining of the double digested and agrarose gel purified vector and insert. This is catalyzed by T4 DNA ligase. At that time foreign DNA was introduced into bacterial cells known as a transformation. Followed by screening for recombinants by separating six colonies and growing them in a 5ml of LB plates. Subsequently purified our recombinant DNA, which is a combination of our vector and insert, to check if our subcloning was successful. We were successful in creating a recombinant DNA.
Sub-cloning of DNA was performed to create a recombinant DNA molecule. We begin by isolating the DNA of choice, the insert, and place it into a vector, also known as a ligation. The vector is then delivered to the host cell and is reproduced along with that host. (Schramm, 7)The DNA that is introduced into the host cell is recombinant because it contains DNA from two different sources. We used the alkaline lysis method to isolate the DNA from bacterial cultures by using SDS detergent to break the cell membranes open and attach to the genetic material. Then performed a restriction digest where restriction endonucleases cut DNA at specific sites. Enzymes: BamHI and XbaI were used to create sticky ends to allow ligation.(Schramm, 9) At that time it was most necessary to purify the restriction digested vector and insert from a 1% agrarose gel. A gel electrophoresis was then performed to determine if they restriction digest worked efficiently. TAE running buffer, a denaturing gradient was then added to the gel. The gel is exposed to an electrical charge, DNA having an overall negative charge moves towards the positive charge. It is separated by size; therefore smaller DNA fragments migrate faster and the larger DNA pieces migrate slower. With the help of Ethidium Bromide when exposed to UV light, makes DNA visible in a agrarose gel. (Schramm, 13)A ligation is then performed where T4 ligase is used to catalyze a joining reaction between DNA molecules involving 3'-hydroxy and the 5' phosphate termini. This enzyme is used to join together the vector and the insert. (Schramm, 17)Ligase requires ATP and Mg++. At that time foreign DNA was introduced into bacterial cells known as a transformation. CaCl2 was used to poke holes in the cell membrane, making the cells "competent". They will be "glued" together and our resulting plasmid is transformed into E.coli so we can select and propagate the recombinant DNA sample (Schramm,17). Three different ratios of vector: insert were made, 1:0, 1:1, and 1:3(Schramm, 18). It is necessary to then screen and purify our recombinant DNA. Six colonies are then chosen (3 from the 1:1 ligation and 3 from the 1:3 ligation) and are grown in 5 ml of LB, containing ampicillin resistant gene (Schramm, 22). Lastly, DNA and restriction digest were isolated to determine whether we have successfully sub cloned our favorite gene, the insert, into our vector, the plasmid. Both the insert and vector have been digested with the same restriction enzyme (HindIII) and subsequently gel purified. I hypothesize that if we properly followed the steps of Sub-cloning techniques then we will be successful in screening for DNA recombinants.
Begin the Alkaline lysis method by pelleting 1.5 ml of fresh bacterial culture at maximum speed for 1 minute in a microfuge tube. Pour of supernatant and make sure any traces of it are removed by inverting over a towel. Repeat this step in the same eppendorf tube, pelleting a total of 3ml. add 400Âµl of cold lysis buffer. Vortex at the highest speed for 30 seconds to obtain the maximum yield. Incubate lysate at room temperature for 3 minutes. Lysate must be poured into labeled spin column, then place spin column into labeled microfuge tube. Centrifuge the spin column for 1 minute at maximum speed; discard the flow through at this point. Add 400 Âµl of wash buffer to the spin column, then centrifuge at maximum speed for 1 minute. Remove tube and decant the flow through the microfuge tube. Return spin column to microfuge tube and centrifuge at maximum speed for 1 minutes to dry the spin column. Transfer spin column to a clean microfuge tube. Add 30 Âµl of water to the center of the spin column membrane, centrifuge at maximum speed for 1 minute. Discard the spin column. The eluted DNA will be quantified and used in a restriction digest.
Now it is necessary to complete a DNA quantification, begin by labeling 3 microfuge tubes, Designate sample 1 as a vector or insert, and sample 2 becomes the remaining sample. Add 1000 Âµl of water to the blank, sample 1 and sample 2 get 98 Âµl water. Add 2 Âµl of appropriate sample to each microfuge tube. Read the absorbance at 260nm and 280 nm from the spectrophotometer. Digest will be incubated at 37°C overnight in two microfuge tubes. They will be frozen and gel purified. The next step Is to isolate and purify restriction digested vector and insert from a 1% agarose gel. To prepare 1% agarose gel place 1gram agarose in an Erlenmeyer flask. Add 100ml 0.5 TAE buffer. Microwave until all is dissolved. Add 10 Âµl of 10 mg/ml Ethidium Bromide (carcinogen), cast gel and allow to solidify. Next to load agarose gel. Add 5 Âµl of 5X gel loading dye to each restriction digest sample prepared from the first part of the experiment. Label a microfuge tube as marker. Add 4 Âµl of 1kb marker and 1 Âµl of 5Xdye. Load samples on gel and record loading order. Split restriction digest as necessary. Run gel at 120 V for 1hour. Photo document. Cut out both insert and vector from the gel. Record the size of insert and vector compared to the 1 kb ladder. Proceed to purification. Purify vector and insert, label each tube, after purifying the DNA samples use 5 Âµl to determine DNA concentration, and record. Use the excise DNA of interest in gel slice and weigh the gel slice. Add 3 volumes of DNA binding buffer for every volume of gel slice. Incubate at 50°C for 10-15 minutes. Add 1X original gel slice volume of isopropanol and mix by inversion of repeated pipetting. Add up to 800 Âµl of sample to the spin column inserted in a microfuge tube. Spin at 10,000Xg for 1 minute. Discard the flow through, repeat if there is any sample remaining. Wash with 750 Âµl of wash buffer. Centrifuge for an additional minute at 10,000Xg. Place spin column in a new microfuge tube and add 20 Âµl water. Centrifuge for 1 minute at 10,000Xg. Next to ligate the double digested and agarose gel purified vector and insert, which then will be transformed into E.coli so we may select and propagate the recombinant DNA.
Begin this by combining 100 ng of vector with either an equal or a 3-fold picomolar excess inert. Adjust volume to 10 Âµl with dH20.add 10 Âµl of 2X ligation buffer and mix. Add 1 Âµl of T4 DNA ligase and thoroughly mix. The mixture should be briefly centrifuged and incubated at room temperature (25°C) for 15 mins. Thaw competent cells on ice, label and transfer to 5 Âµl of ligation mixture to a 1.5 ml microfuge tube. Add 100 Âµl of competent cells to the DNA and gently mix through pipetting up and down. Incubate on ice for 30 minutes. Heat shock for two mins at 37°C, chill on ice for 5 mins. Add 850 Âµl of Luria Broth (LB) and incubate at 37°C for 1 hr. continue and spread 200 Âµl onto the appropriate LB plates with the correct antibiotic to select for recombinant DNA. Incubate overnight at 37°C, and record the number of growth colonies. It is important to now screen for recombinant by indentifying and purifying our recombinant DNA.
DNA isolation and purification must begin by pelleting 1.5ml of fresh bacterial culture at maximum for 1 minute into a 2ml culture tube. Pour off supernatant; do not disturb the bacterial pellet. Repeat in the same microfuge tube. Add 400Âµl of cold lysis buffer, vortex thoroughly at high speed for 30 seconds. Incubate at room temperature for 3 mins. Transfer and decant into a labeled spin column in a microfuge tube. Centrifuge spin column for 1 minute at maximum speed. Remove the spin column and decant the filtrate, place spin column back into microfuge tube in centrifuge. Dry it by running a maximum speed. Transfer spin column to a labeled 1.5ml microfuge tube. Add 30Âµl of water to the center of spin column membrane, avoid contact. Incubate at room temperature for 2 mins. Centrifuge at maximum speed for 1 min. remove and discard the spin column. Eluted DNA is used in a restriction digest. Begin restriction digest of mini prep DNA by labeling 4 tubes, and an additional tube MM-master mix. Add 1Âµg of each mini prep to a labeld tube, add water so the final volume is now 7Âµl in each tube. Add 3Âµl of MM to each and briefly spin. Incubate at 37°C for 30 mins. Add 2Âµl of 5X dye to each sample and run on 1% agarose gel.
Overall, our findings were successful in forming recombinant DNA. For the first part of the experiment, we isolate and purify plasmid DNA and restriction digest both insert and vector. The concentration values for the insert and vector were too low, meaning protein contamination. In Sub-cloning II, a gel purification of restriction digested vector and insert was run, as seen in figure 1. In Sub-cloning III: Ligation and Transformation of the double digested and agarose gel purified vector and insert, as seen in figure 2, three plates 1:0, 1:1, 1:3 ratios of insert: vector are shown. Growth colonies increase from 1:0 to 1:3. In screening for recombinants, we ran a HindIII digest and have 2bands.
Overall I fully support my hypothesis; our finding in the sub-cloning experiment was successful in that we created recombinant DNA. In sub-cloning I we isolated and purified plasmid DNA from E.coli and performed a restriction digest both insert and vector for directional sub-cloning experiment. We were not able to use our own prepared vector and insert due to the fact that we had some accidental spillage from our eppendorf tubes and our concentration values were very low, as seen in Table 1. Therefore we had to use the prepared samples provided to us from our TA's. In sub-cloning II we isolated and purified the restriction digested vector and insert from a 1% agarose gel that was prepared by our TA's. We inserted our insert and vector into the electrophoresis gel and then incubated for about 1 hour. After incubation our insert fell between tracks 2 and 3 and our vector fell between tracks 6 and 7 (figure 1). We then cut the insert out of the gel and put it into a microfuge tube and then cut out the vector and put it into a different microfuge tube. Our insert had a high ratio of 3.97; it was a more pure sample meaning it had RNA contamination, as seen in table 2. Our Vector had insanely low numbers, meaning there was protein contamination (table 2), therefore we were not able to use our own insert and vector and had to use those provided by our TA's. In Sub-cloning III we were unable to do this experiment due to a snow day, we again used Dr. Schramm's prepared bacteria of E. coli so we can select and propagate the recombinant DNA. There were three plates used: Our 1:0 ratio of insert and vector is our control to known if our experiment is going well, and to check for undigested vector. Since we had minimal growth on the plate we had no undigested vector (figure 2). The 1:1 plate had the same amount of insert and vector, and we had a decent amount of growth (figure 2). The 1:3 plate had 3X more insert than vector and had the most growth of bacteria (figure 2). Therefore Dr. Schramm's ligation and transformation was successful. In Sub-cloning IV for screened for recombinants by identifying and purifying our recombinant DNA(figure 3) This last sub-cloning procedure concluded our experiment. As a result, we were successful in creating recombinant DNA. In Sub cloning IV: Screening for recombinants we used the restriction enzyme HindIII to cut both vector and insert. Two bands showed on our electrophoresis gel meaning it was successful.
From our Sub-cloning experiment I have successfully learned how to create recombinant DNA. I learned how to create recombinant DNA by productively isolating and purifying DNA and the restriction digest, then isolating and purifying it from 1% agarose gel, followed by ligation of the double digested and agarose gel purified vector and insert, which was transformed into E.coli, followed by screening for the recombinant DNA
Schramm, Laura. Molecular & Cellular Biology Laboratory. New York: Bent Tree Press, 2009.
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