Restriction Digest Involves The Uses Of Restriction Enzymes

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

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

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).

We were using a restrictive enzyme to cut the DNA into smaller fragments. For the restriction digest we pipet 4 micrometers of enzyme mix into the bottom of each of our colored tubes making sure to use a new tip for each sample. Next, we capped the tubes and mixed the contents by flicking the tube a little bit with our fingers. After we mixed the contents, we tapped the tube to make sure all the liquid would go to the bottom of the tube. Then, we put the colored tubes in the heating blocks and we let them incubate for 35 minutes at 37 degrees Celsius.

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.

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)

(2012) introduced a new genetic technique that was derived from the defense mechanisms of bacteria. Some bacteria use a CRISPR-Cas system to defend against foreign viral and plasmid genetic material. Once foreign targets enter the system, the bacteria will integrate its CRISPR array to parts of the nucleotide sequences on the invading sequence. The bacteria will then produce a precursor CRISPR-RNA that complements the invading sequence, and is used to find all foreign sequences that match it. These precursor RNAs will work with Cas proteins to cleave the foreign sequence, thus effectively silencing it. There are multiple types of CRISPR-Cas systems that bacteria use. Type 2 systems, paired with Cas-9, use another RNA sequence, tracrRNA (trRNA), as a complement to precursor CRISPR-RNA. These systems used both trRNA and precursor CRISPR-RNA to induce a double stranded cleave. After this discovery, a Cas9 protein was purified and tested to see if it would be able to cleave DNA. It was discovered that if both a trRNA and a precursor CRISPR RNA were present with complementary sequences to a sequence in a DNA strand, the result would be a double strand cleave in the DNA. Cas9 also contains two domains, each of which only cleave either the complementary or the non-complementary strand of the target DNA. After looking at both the trRNA and the precursor CRISPR RNA, researchers theorized that they could engineer a chimera RNA that combined certain sequences of both

This strategy can be employed to study enzymes from other Gram-positive organisms with ease. It

The chromosomal DNA was digested with different dilutions of Bam H1 (10units/µl). The reaction mixture consisted of 10 g of chromosomal DNA and 10 l of the restriction enzyme buffer in a final volume of 100 l. In the first PCR tube 20 l of the reaction mixture was dispensed while 10 l was dispensed in 2 to 7 PCR tubes. The tubes were placed on ice for 2 mins and 10 units of the Bam H1 were added to the first tube. The contents were mixed and 5 l from the first tube was transferred to the second tube.

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

Within a genome, there is a vast sequence of DNA that may be studied. The resulting goal of this study is to create a genomic library of the bacteria Aliivibrio Fischeri. We will be achieving this purpose by making Escherichia Coli luminescence through the use of the lux operon. In the process of understanding the genomic library of A. Fischeri bacteria, we will be creating a restriction map of the restriction sites in the plasmids containing a lux.

Did you know that with the science of DNA manipulation, animal cadavers can be turned into insulin for diabetics? Back in the 80's scientists isolated the human gene for insulin and transferred it into bacteria. Now bacteria cultures are used to produce large amounts of human insulin. DNA manipulation is especially important in medicine, where it holds the hope of curing genetic diseases such as Huntington's, and even some types of cancer. There are several major types of DNA-manipulation enzymes used by living cells. The first type is DNA polymerase, which cells use to replicate their own DNA. The next type is DNA ligase, which joins 2 pieces of DNA to create a single piece. The third type is restriction enzymes, which appear to be made only by bacteria. Restriction enzymes are very important enzymes that are vital to our manipulation of DNA (Rapoza, DNA, 2).

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

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