Restriction Digest

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).

Find out more about restriction enzymes by viewing the animation and reading the article listed below.

We placed the gel into the running chamber, and then we completely covered the gel with TAE. 3 microliters of loading dye was added to each tube; this would help distinguish the enzyme from the gel. As before, we tapped the tube on the table to mix. Then we carefully added each of the four samples into their own wells. A total of 33 microliters of each sample was poured into each well. Afterwards, we attached the positive and negative electrodes to their corresponding terminals on the power supply and gel box. We turned on the power to around 80 volts and waited 45-60 minutes for the loading dye to move down the gel approximately 6-8 cm. Finally, we were able to visualize the DNA in the gel and write down the

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

 By restriction enzymes then amplified by polymerase chain reaction to make many to millions of copies of a single fragment.

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

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)

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.

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

Enzymes are applied to DNA to break it into smaller pieces which are called restriction endonucleases. These restriction endonucleases become

We use eight lanes putting the enzymes, and the DNA of the suspects to the lane. It is important to do this slowly and with care so as to ensure we put each solution in the correct tubes. You also have to plug it to the power source. Red is the negative while black is the positive and it should be at 150V for about forty minutes. Evaluation of the DNA bands

Enzymes are very specific in nature, which helps them in reactions. When an enzyme recognizes its specific substrate, the

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double

The physical work of genetic engineering couldn’t have been done without the tools of biotechnology i.e. enzymes that cut he DNA molecules in order to select the wanted/ remove the superfluous genes. These specific enzymes came to be known as restriction enzymes. In nature it needs to be able to recognize and cut very specific factions of foreign DNA therefore protecting the bacteria against the unknown substance in a process called restriction. The bacterial cell then continues to protect its own DNA from restriction by adding –CH3 to adenines or cytosines within making the strand recognizable by the restriction enzyme as to avoid being cut. In biotechnology EcoRI acts as our restriction enzyme and makes a cut into the sugar phosphate backbone of the DNA in a section it can recognize. The complementary

Scientists by nature are endlessly fascinated with the unknown and, consequently, with harnessing new scientific techniques for the ongoing benefit of society. One example of this is the restriction enzyme, which is believed to have first been discovered in the 1950s by Salvador Luria and Mary Human, but then undergone further research in the following decades (Pray, 2008). The application of the restriction enzyme can be described as the ability to cut sequences of DNA at specific regions. When the discovery of the restriction enzyme occurred it was ground breaking, however, fast forward to the present day and it seems as though science has far surpassed this achievement with the serendipitous discovery of an even more beneficial technique