Preview

2x Rapid Ligation Lab Report

Good Essays
Open Document
Open Document
468 Words
Grammar
Grammar
Plagiarism
Plagiarism
Writing
Writing
Score
Score
2x Rapid Ligation Lab Report
Ligation is the process of joining two DNA fragments or other molecules by a phosphate ester linkage with the action of enzyme. Ligation of DNA is process that involve the DNA of interest is inserted into the plasmid. This 3’-hydroxyl of DNA terminus are joined together with 5’-phosphoryl of another by phosphodiester bond. The ligation of DNA fragments usually performed by using T4 DNA ligase. All the reaction components such as ligation buffer, DNA insert, pGEM®-T, and T4 DNA ligase is mixed by gentle pipetting. The 2X rapid ligation buffer is used in this experiment to saving the ligation time. This ligation buffer contains ATP. ATP is used to catalyze the formation of phosphodiester bond and the reaction of restriction enzyme buffer. The recommended time and temperature of incubation when using 2X rapid ligation buffer are one hour at room temperature (24°C) and overnight at 4°C. In this overnight incubation at 4°C is applied to achieve maximum number of recombinants. …show more content…
The single 3’-T overhangs which usually called as “sticky ends” at the insertion sites helps to increase the ligation of DNA fragments into plasmid by preventing recircularization of vector. When the overhang is compatible with the base pairs, the two fragment of DNA will connect by ligation reaction. The new pGEM®-T and pGEM®-T Easy Vector Systems already contain the 2X rapid ligation buffer so that T4 DNA ligase can be used for one-hour ligation reactions instead of overnight ligation. T4 DNA ligase is used to catalyze the joining between 5’ phosphate and 3’ hydroxyl groups of adjacent nucleotides. The DNA ends can be cohesive ends or blunt ends configuration. Ligase usually performed at 4°C overnight or one hour at room temperature

You May Also Find These Documents Helpful

  • Better Essays

    the foreign DNA successfully inserted can be easily identified from the nonrecombinants based on the colour difference of colonies on growth media.…

    • 1878 Words
    • 27 Pages
    Better Essays
  • Better Essays

    Nt1310 Unit 1 Exercise 1

    • 1475 Words
    • 6 Pages

    Denaturation is carried on by heating the double-stranded DNA at 94°C to separate the complementary strands that will serve as template in further cyclings. Pre-denaturation is sometimes done at the same temperature to ensure complete separation of strands. Annealing then occurs upon rapid cooling of the solution, allowing oligonucleotide primers to hybridize to the template. In this phase, however, the single strands of the template are too long and complex to be able to completely reanneal spontaneously. The gene fragment to be amplified will completely form double-stranded fragments upon further cycling of this step and the extension step. The extension step involves heating of the reannealed DNA to 72°C, the temperature at which the thermostable DNA polymerase in the mix will operate most efficiently in synthesizing new DNA strands.…

    • 1475 Words
    • 6 Pages
    Better Essays
  • Good Essays

    This was done by melting a solution composed of 0.7% agarose in 1x TBE and 0.005% ethidium bromide, and pouring the resulting liquid into comb-containing gel tray until the thickness reached about 6 mm. Once the gel had been allowed to cool for about 20 minutes, it was removed from the tray, placed in a horizontal gel apparatus, and immersed in 1x TBE buffer. Next, three different solutions were prepared from an aliquot of HindII digested lambda DNA and 3 microliters of 6x loading dye, an aliquot of EcoRI digested lambda DNA and 3 microliters of 6x loading dye, and 1 kb DNA ladder and 3 microliters of 6x loading dye. The resulting solutions were then transferred into separate wells within the gel, and the apparatus was connected to a constant current source (45mA) for 60 minutes. After producing clearly visible bands, the gel was taken out of the apparatus and photographed with an ultraviolet light box (Displayed in Figure…

    • 1568 Words
    • 7 Pages
    Good Essays
  • Good Essays

    D) cut the plasmid twice with restriction enzyme Y and ligate the two fragments onto the ends of…

    • 4889 Words
    • 20 Pages
    Good Essays
  • Powerful Essays

    Sq3r Chapter 13

    • 1466 Words
    • 6 Pages

    6) Recombinant DNA is created from joining two different fragments. In the process of studying recombinant DNA, large amounts of recombinant DNA are needed. The recombinant DNA is transferred into the bacterium through a carrier/vector. Plasmids and viruses are commonly used vectors. An enzyme, DNA ligase, joins the 2 DNA fragments chemically.…

    • 1466 Words
    • 6 Pages
    Powerful Essays
  • Better Essays

    4. Cut plasmid and eukaryotic DNA with the same restriction enzyme… Mix fragments and allow them to match… Ligate… Transform into bacteria… Identify desired clone.…

    • 1001 Words
    • 5 Pages
    Better Essays
  • Satisfactory Essays

    Microbiology Task 1

    • 406 Words
    • 2 Pages

    Ligase is the linking enzyme that seals breaks in the DNA by creating a phosphate-sugar bond. DNA ligase has three (3) main functions: 1. joining Okazaki fragments, 2. sealing repairs, 3. sealing recombination fragments…

    • 406 Words
    • 2 Pages
    Satisfactory Essays
  • Good Essays

    Gene Transfer Lab Report

    • 939 Words
    • 4 Pages

    The following experiment method is based on the procedure given through the Biology Department at UWM (Wimpee, 2006). This experiments started with two tubes of 100 uL E. coli cells, labeled one and two. Tube one just contained normal E. coli cells. Tube two was the tube with the plasmid added to it. The first step in this experiment was to add plasmid DNA, the “mini chromosomes” of the bacteria, to the E. coli cells in order to change the genetic makeup of them. I then added 10 uL of the plasmid to tube two. The next step was to chill both the tubes E.…

    • 939 Words
    • 4 Pages
    Good Essays
  • Good Essays

    Biology

    • 696 Words
    • 3 Pages

    1. Outline the process of DNA profiling (genetic fingerprinting), including ways in which it can be used. 6 marks…

    • 696 Words
    • 3 Pages
    Good Essays
  • Satisfactory Essays

    Biology Meiosis Mitosis

    • 491 Words
    • 2 Pages

    First: The parental strand divides in 2 separate strands; the helicase unwinds by cutting hydrogen bonds. Then, each strand is a template that attracts and binds complementary nucleotides, meaning that each daughter strand will bind proteins to the half to keep the DNA molecule separated. DNA polymerase attaches to DNA nucleotides, and it assembles nucleotides to the half existing strand. Ligase (an enzyme) forms covalent bonds between adjacent segments of the newly created DNA strands stitching up the gaps. Last, it re-winds the helix. (Gyrase)…

    • 491 Words
    • 2 Pages
    Satisfactory Essays
  • Good Essays

    Gene Splicing

    • 372 Words
    • 2 Pages

    Chemicals called restriction enzymes are used as scissors to cut the DNA. Thousands of these restriction enzymes exist, each recognizing only a single sequence of DNA. Once it finds that sequence in a strand of DNA, it attacks it and splits the base pairs apart, leaving it like a broken chain. Scientists can now add any DNA they wish to this “broken chain” and, afterwards, the chain is repaired with an enzyme called ligase.…

    • 372 Words
    • 2 Pages
    Good Essays
  • Good Essays

    Dna Reproductive Process

    • 1062 Words
    • 5 Pages

    When the two strands of DNA double helix are separated, each can serve as a template for the replication of a new complementary strand, producing two daughter molecules each of which contains two DNA strands with an antiparallel orientation. The enzymes involved in DNA replication process are template-directed polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity. This complex leads to the local denaturation and unwinding of an adjacent A + T rich region of DNA. The interaction of proteins with the origin is what defines the start site of replication and provides a short region of single stranded DNA essential to initiation of synthesis of the nascent DNA strand. Then helicase binds and allows for processive unwinding of double stranded DNA into single stranded DNA. As helicase unwinds the DNA, DNA single stranded protiens bind and stabilize the single stranded DNA. The polymerase III holoenzyme binds to template DNA as a part of a multi protein complex that consists of several polymerase accessory factors. DNA polymerase synthesizes DNA only in the 5 ' to 3 ' direction and only one of the several different types of polymerases is involved at the replication fork. As the DNA strands are anti parallel, the DNA polymerase functions asymmetrically. On the leading (forward) strand, the DNA is synthesized continuously. On the lagging strand (retro strand) the DNA is synthesized in short (1-5 kb) fragments. These DNA fragments are called as okazaki fragments. The proof function identifies copying errors and corrects them. Polymerase III is an enzyme with high processivity and catalysing capacity than others. The initiation of DNA synthesis requires priming by a short length of RNA about 10-200 nucleotides long. This priming process involves the nucleophilic attack by the 3' -OH group of the RNA primer on the aphosphate of the deoxyribonucleotide triphosphate that enters first,…

    • 1062 Words
    • 5 Pages
    Good Essays
  • Powerful Essays

    10. QIAGEN. 2006. Protocol: Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge. QIAGEN QIAprep Miniprep Handbook 2nd edition. 22-23.…

    • 3652 Words
    • 15 Pages
    Powerful Essays
  • Good Essays

    pretty

    • 1321 Words
    • 4 Pages

    The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.…

    • 1321 Words
    • 4 Pages
    Good Essays
  • Powerful Essays

    Polymerase Chain Reaction

    • 4301 Words
    • 18 Pages

    The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.…

    • 4301 Words
    • 18 Pages
    Powerful Essays