Locating the XhoI Recognition Site on Lambda DNA Using a HindIII and XhoI double digest
Haleigh Wood
Abstract
Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA
…show more content…
XhoI is a type I restriction enzyme, so the location of the recognition site on lambda DNA is unknown but it is known that it cleaves at CTCGAG sites (XhoI (10u/ul)). While the DNA fragment lengths and cleavage sites are known for HindIII lambda DNA digest, they are not known for a XhoI lambda DNA digest. The goal of this study is to determine the recognition site of XhoI on lambda DNA by comparing the DNA fragments from a HindIII digest to the DNA fragments of a HindIII and XhoI digest. We will do this by creating a mixture of solutions containing only DNA, DNA and HindIII, DNA and XhoI, and DNA, HindIII and XhoI. We will then run a gel electrophoresis, which will separate the DNA, fragments by size and we will compare the DNA fragments from each solution. Through comparing the DNA fragments from the HindIII digest and the HindIII and XhoI double digest, we will be able to determine the XhoI recognition site on the lambda DNA.
Methods
Preparation of restriction digest solutions:
To begin the process to determine the XhoI recognition site in the lamda DNA fragment we first prepared 4 tubes of solutions containing 10X Optizyme reaction buffer, sterile water, lambda DNA (0.3 ug/ul), XhoI (10u/ul, 3000u), and HindIII (10u/ul, 7500ul). Tube 1 contained 2ul 10X Optizyme, 16ul sterile water, and 2ul lambda DNA. Tube 2 contained 2ul 10X Optizyme, 14ul sterile water, 2ul lambda DNA, and 2ul XhoI. Tube 3 contained 2ul 10X Optizyme, 14ul sterile water, 2ul
Restriction enzymes are a tool that allows us to pinpoint human identity down to single differences in our DNA. Work through the following simulation so you can see these molecular scissors in action.
The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).
The following results helped obtain the haplogroup that in which the sequence of mtDNA would identify. The PCR reaction worked, and this can be determined by looking at the agarose gel in figure 1. If the PCR reaction was successful, than a band should appear around 550bp. Individual AC displays a band around 550bp, this means the PCR reaction was successful. The band for individual AC, depicts a low concentration of product, because the band faint. After the purification process the concentration, A260/280 ratio, and A260/A230 ratio were determined by using the nanodrop. The concentration of mtDNA in the product was 60.9 ng/uL. The ratio for A260/280 was 1.79 and the ratio for A260/230 was 0.77. The A260 and 280 are a spectrometer measurement that measure absorbance at wavelengths of
How to determine where the restriction enzymes, Ava II and Pvu II, sliced the DNA.
Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific
Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.
The first agarose gel provided insight into whether or not Zassy’s genomic DNA had been cut by the Sal I HF (NEB) restriction enzyme. As seen in Figure 1, Zassy’s cut DNA, in lane 3 left clear fragments between the 3 kb and 0.5 kb areas. Zassy’s uncut DNA, in lane 2, left a clear and bright band above the 10 kb marker. Therefore, the gel confirmed that Zassy’s DNA was successfully cut with Sal I because there was a significant difference the cut and uncut DNA that was run through the gel. There was an additional lane of cut DNA because another student adopted Zassy in the early stages of this experiment. Nevertheless, the clear distinction between lanes 2 and 3 allowed the experiment to continue on to purification of the DNA.
All the procedures performed were according to the Biochemical Techniques: Recombinant DNA Laboratory Second Edition (1). Restriction Digestion of Unknown Vector An unknown vector (0.1 µg/µL) was assigned to the group for restriction enzyme digestions. The vector could be either pUWL 500 or pUWL 501.Three enzymes, Pst I, Bgl II, and Eco RV, and their combinations were used to single, double and triple digest the unknown vector. A total of nine tubes of digestion mixtures were made for this experiment.
The gel was covered with a buffer and then six samples labeled A-F were deposited into the wells using a micropipette. Three of these samples (A, B, C) were control samples to compare to D, E, and F (the mother, child, and father). The safety cover was placed on the unit and then brought to a power source. The leads were connected the chamber and left for approximately 20 minutes. The agarose gel was removed from the tray and placed onto a sheet of plastic wrap. An Ethidium Bromide card was placed face down onto the gel to stain for approximately 5 minutes. Finally, the card was removed and the gel was placed on top of a UV light. The samples were pushed towards the center due to opposite electric charges. Agarose gel separates the DNA samples by the way they were cut. The restriction enzyme MST II cuts the DNA strand at CC/TNAGG where N is any nucleotide base. If the enzyme recognizes this, it is cut. If it does not recognize it then the strand is left whole. We were able to observe the DNA strands due to them being dyed and placed over a UV light. The control samples were utilized so that the other samples could be compared to test for their genotype. The data was analyzed in this way to differentiate between the different genotypes and the number of bars they
The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.
Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)
In Southern blotting DNA is extracted, purified, and cut into fragments with restriction enzymes. The DNA fragments are separated by size using gel electrophoresis. The DNA is transferred from the gel to a nitrocellulose filter by placing the gel on top of a sponge sitting in a tray filled with buffer. A nitrocellulose filter is laid over the gel and covered with paper towels. As the paper towels pull the buffer through the sponge, gel, and filter the DNA fragments are carried from the gel to the nitrocellulose filter where they stick tightly. The nitrocellulose filter is removed and hybridized with a radioactively labeled nucleic acid probe that tags the DNA fragments of interest. Unbound probe is washed off and the filter is exposed to X-ray film. The DNA fragments that are
On the other hand, using genomic characterization and restriction site mapping of HN, F and L
ot laed retteb a noi tisiuqca eht gnikam ,n oillim 927 $ ot n oillim 959$ m orf IKB fo eulaV esirpretnE nac reriuqca eht esuaceb srevoekat elitsoh tcartta lliw sei tiruces dna hsac 132$ ehT – taerhT revoekaT lareves evah y ticapac tbed dna level hsac sulprus hgiH .tbed on htiw teehs ecnalab sti no gnittis .IKB
The chemical and reagents used for the extraction and quantitation of DNA were: Plant DNAzol (0.3ml/0.1g), 100% ethanol (100%: 0.225 ml/0.1 g, 75%: 0.3 ml/0.1 g), Chloroform (0.3 ml/0.1 g), Plant DNAzol-ethanol solution: Plant DNAzol, 100% ethanol (1:0.75 v/v), TE buffer (10 mM Tris, 1 mM EDTA pH 8.0), 1.2% agarose gel (Agarose, 1X TAE buffer), 6X loading buffer (glycerol, Tris/EDTA pH 8.0, ethidium bromide), .25X TAE buffer, Restriction enzymes and Restriction endonuclease buffers. All the chemicals used were quality grade. The restriction