Restriction enzymes and DNA ligase are very important parts in theprocess of molecular cloning. In this experiment, Lambda DNA was digested using restriction enzyme HindIII and then ligated back together using DNA ligase. The partially ligated DNA and the fully ligated DNA were compared to the original DNA side by side on an agarose gel. The results showed that a significant amount of Lambda DNA was digested and the ligation stages had visible progress.
Introduction Restriction enzymes (restriction endonuclease) are enzymes that cut DNA at a specific region of nucleotide sequences, known as the restriction site. To cut the DNA, restriction enzymes make two incisions, one through each strand, or sugar-phosphate backbone. After cutting, another
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On the side, another batch of lambda DNA (1.68 µL) was mixed with nuclease free water (3.32 µL).
During the incubation, in an Erlenmeyer flask, 1X Tris Acetate EDTA (1 mL) and powder agarose (0.4 g) were dissolved in dH2O (49 mL). Then the solution was microwave for 2 minutes and allowed to cool to room temperature. Then SafeRed concentrate (2.5 µL) was added to the solution and it was poured into the gel box and allowed to solidify.
After the incubation, the sample was heated at 80 °C for 20 minutes to inactivate HindIII, then cooled to room temperature. During this step, two “stop” tubes labeled 1’ and 10’ each containing 50 mM EDTA (5 µL) were prepared. After the inactivation, the DNA solution (5 µL) was transferred into another tube labeled “cut”. For the remaining DNA, 1X final ligase buffer (2.5 µL), T4 DNA ligase (1 µL), and nuclease free water (6.5 µL) were added and incubated at room temperature. After 1 minute, the solution (10 µL) was transferred to the 1’ stop tube. After 10 minutes, the remaining solution (10 µL) was transferred to the 10’ stop tube. Finally, DNA gel-loading buffer (3 µL) was added to the “cut” tube, 1’ stop tube, and 10’ stop
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From the cut DNA lane, the digestion reaction worked, as evidenced by several visible bands in that lane, but it did not go to 100% completion. The 1’ stop lane has largely one band, but it also has several faint bands, which means that the DNA fragment has begun to ligase back together. In the 10’ stop lane, there is one band, which means that the fragments are largely ligated back together.
Summary and Discussion The restriction digest did not go to completion because the top band is very large and almost the same size as the uncut DNA, which means that some DNA fragments have been digested and most of them are still intact. Also there are no bands at the bottom of the lane because fragments are very large by themselves. In the future, this can be improved by allowing more time to digest during incubating and heating, or using enzymes that have 100% activity instead of enzymes that were stored for a long time. The band pattern seemed to match the calculated, but have some bands supposedly missing, according to the Table 1 below. On the gel, there is a faint band at ~700 bp, a band at ~2000 bp, a band at ~4500 bp, and a band at ~7000 bp. These bands correspond to #7, #6, #4, and #3, respectively on the left table.
Table 1. Predicted band pattern when the lambda DNA is cut by
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
The product of the reaction of the A primer seems to have failed as no bands were produced apart from the terminal point of the migration which is too small to be considered as either a preintegration site or a retrovirus containing section, not only did my partner seem to have the same problem, most if not all of the submitted gels seem to have no bands for the A set of primers.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
The main goal for our experiment was to learn how to examine DNA when there is only a small
This experiment requires four tubes with an enzyme solution, chelating agent and deionized water. Also a fifth tube that is the calibration tube for the spectrophotometer, which only has 5ml of dH2O. The calibration tube is used to level out the spectrophotometer to zero before each trial. The spectrophotometer was set at 540 nm, “since green is not a color seen with the conversion of catechol to benzoquinone.” The enzyme solution was made by using potato that was peeled so that the golden color of the skin wouldn’t react or interfere with the red color needed in the spectrophotometer. After it was peeled, it was cut into chunks to minimize excess heat created while it was blended. It was put in a chilled blender and 500ml of deionized water was added. Chilled, deionized water was used because it created a hypotonic environment that caused the cells from the potato to burst and release the catecholase. It was chilled
A helicase uses energy provided by ATP to uncoil the DNA template specified (Biology pg. 267). The helicase essentially divides the DNA, so that it can be able to form a replication fork in its origin of replication (Biology pg. 268). Then, Okazaki fragments are formed in the lagging strand. Okazaki fragments are defined as “DNA fragments synthesized on the lagging strand (Biology pg. 268).” Meanwhile, the leading strand is still continuously replicating (Biology pg. 268). After the lagging strand synthesis, which is when “the primase synthesizes the primers needed by DNA polymerase III”, the DNA ligase closes the gaps between the Okazaki fragments (Biology pg. 268-269). Finally, termination occurs at an opposite spot of the origin. In the final stage two daughter molecules are produced and are interlocked in a chain-like
Structural genes code for the enzymes themselves. RNA polymerase transcribes all of the genes into a polycistronic mRNA. The promoter is the site where the RNA polymerase binds to the DNA prior to beginning transcription. The operator serves as the binding site for the protein called the repressor. The regulatory gene encodes the repressor protein.
A Ponceau stain can bind and identify all proteins. Lanes 2, 3, and 4 (our recombinant, nonrecombinant and green colony, respectively) have a slightly smeared pattern of multiple bands that goes from 245 kDa to 80 kDa. Lanes 2 and 4 have faint banding patterns that descend from 80 kDa downwards. Lane 3 ends a bit early, around the 135 kDa mark. Lanes 5-7 (our white colony, unknown colony and purified
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
Use techniques such as electrophoresis and restriction mapping to understand the concepts of DNA digestion. Also, become familiarized with restriction enzymes and double digestion.
Uncut vector alone: shows if the transformation works. If positive, many colonies would appear. 2) Cut vector alone without ligation would result in no colonies. If they appear, it means that the vector was not
It is used in many labs and only requires the DNA in question, primers that anneal to the beginning and end of the target genes, Thermus aquaticus, Taq DNA, a heat stable DNA polymerase and all four of the deoxyribonucleate triphosphates. There are three steps in the PCR reaction denaturation, hybridization and DNA synthesis. During these steps the DNA is separated or denatured into two strands, hybridized, where the two single strands are complimentary paired to the respective primers, and then the DNA is synthesized with Taq DNA. This is considered one cycle, and it can commonly take 50 cycles to amplify enough DNA to be used. When the PCR is completed a gel electrophoresis is run. The PCR product is put in a specially formed agarose gel that will allow electricity to flow around the gel and DNA and force the DNA to travel down the gel resulting in white bands depending on their electronegativity. When the DNA is transformed from plasmid into the yeast we use salmon sperm to protect the nucleus from becoming degraded and the plasmid lost. This increases the efficiency of the DNA because the sperm DNA will adhere to the yeast cell wall and allow the plasmid to bind to the
The restriction enzymes SmaI cuts DNA vertically. This results in two DNA fragments with blunt ends. Next, the gene is spliced into a vect... ... middle of paper ... ... le by stopping illness but this process has also been vandalised for many uses which are not necessary.
When the enzyme finds one of these sequences, it severes the DNA by catalyzing the hydrolysis of the bonds holding adjacent nucleotides together in a process called enzyme digestion. If the DNA is double stranded the sequence is on both strands and runs in opposite directions, allowing the restriction enzyme to cut both strands. There are two possibilities when it comes to cutting double stranded DNA: a blunt cut, where the ends severed are even, or a sticky end, where one end is longer than the other and has a string of nucleotides dangling over the other strand. Bacteria protect themselves from restriction enzymes by disguising their recognition sequences through the methylation of the sequence’s Adenine or Cytosine bases. In molecular biology, restriction enzymes can be used to manipulate DNA fragments and are extremely important tools for recombinant DNA technology and genetic