biotechnology lab class :1. The bacterial streaking on antibiotic containing agar plates has been completed and the plates will be available for you to inspect in the lab class.2. You will complete a restriction enzyme digest on the four available plasmid stocks and then examine the products by agarose gel electrophoresis.Photographs of the gel are attached to identify the plasmid present in each analysed sample.Now consider the following questions :- For each of the four plasmids used in the practical, identify the size (in kb) of the linear DNA fragment(s) that you would expect to obtain if the EcoRI digest is complete.- Using the provided negative image of the gel, which shows the bands present in the ND and D samples for each plasmid, identify the plasmid that was present in each of the four plasmid stock.- Explain how you were able to identify plasmid in each sample using the gel, considering how linear DNA fragments of different length are separated by agarose gel electrophoresis.- Why do supercoiled, nicked and linear DNA sequences of the same size (kb) migrate at different rates during agarose gel electrophoresis?- How does SYBR Safe® enable the visualisation of the location of DNA fragments on the gel? PART TWO: AGAROSE GEL ELECTROPHORESIS OF PLASMID DNA SAMPLESMaterials6x agarose gel loading dyeSterile distilled water (dH₂O)ⓇTBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0, 1x SYBR Safe dye)Pre-prepared 0.8% agarose gel (8 g/L agarose in TBE buffer) containing SYBR Safedye1 kb marker ladder of linear DNA fragments (1 to 12 kb)MethodLane 1DNAladderLane 2Plasmid 1"ND"Lane 3Plasmid 1"D"Lane 4Plasmid 2"ND"Lane 5Plasmid 2"D"ND1. Confirm that the sample from above has not evaporated. If the sample has evaporated significantly, itcan be made up to 10 μL with dH₂O using a P20 pipette, gently mixing for 5 min to ensure that any DNAdried onto the tube has dissolved. Prepare Part 2 samples by adding 1.6 μL only of 6x agarose gel loadingdye to both ND and D. Centrifuge for 10 s to ensure all liquid is at the bottom of the tube.2. The gels and buffer contain SYBR Safe dye - a less mutagenic and safer alternative to ethidiumbromide. Take care to dispose of P200 tips into the biohazard bags immediately after use. There will be ademonstration of how to load the gel with 1 μL the 1 kb marker ladder sample. Load 8 μL of the "D" and"ND" samples into adjacent sample wells in the gel in the order ND, D.#3Lane 6Plasmid 3"ND"DLane 7Plasmid 3"D"#2ND D-10.0 42-8.0 42Lane 8Plasmid 4"ND"-6.0-5.0-4.0# 1-3.0#4504233-1.5125-2.0 483. The identity of all samples must be clearly indicated as this will be required to interpret the results later.The well in which the marker ladder has been loaded must also be indicated. Note that there will be 10 µLof sample in your ND and D tubes but only hold 8 μL is loaded into wells.ND D ND4. When all samples are loaded, the gel electrophoresis should be run at 60 volts until the fragments havemigrated a sufficient distance.36What is the overall charge on the DNA fragments? Towards which electrode will they migrate?-1.0 425. In order to visualise the DNA, the gel contains SYBR Safe dye. The distance that the different sizedmolecules of DNA have migrated can be seen using a blue-light box. You can see the progress of themigration throughout the practical class.Model Data-0.5 42Lane 9Plasmid 4"D" Materials• Plasmid DNA sample at a concentration of 100 ng/µL in sterile, nuclease-free water, labelled "P"ECOR1 restriction enzyme10x concentrated restriction enzyme buffer·• Nuclease-free waterMethod1. Using P20 pipette, transfer 2.5 µL of the purified plasmid "P" into each of two fresh tubes. One labelled"ND" (for non-digested) and the other labelled "D" (for digested). Treat each tube as follows:• To the "ND" tube add 1 μL concentrated restriction endonuclease buffer and mix gently. Then add6.5 μL dH₂O, mix gently. Close the lid.• To the "D" tube add 1 µL restriction endonuclease buffer, mix gently. Then add 1 μL of EcoRIrestriction endonuclease enzyme. Then add 5.5 µL dH₂O, mix gently. Mix gently. Close the lid. Besure to close the lids of both tubes properly to avoid evaporation when at 37 °C and note thevolume.2. Place both "D" and "ND" tubes in the tube rack in the thermostatic block to incubate at 37 °C for 20minutes. During this time the digestion of plasmid DNA will happen. Depending upon the plasmid, thecircular plasmid should be cut at one or two sites generating staggered (sticky) EcoRI ends.Look now again at the plasmid sequences above and identify the EcoRI recognition site(s).3. Check that the samples ...sample is not at the bottom of the tube.Draw a simple diagram of the four plasmids showing the EcoRI recognition site(s). Think about thelinear DNA products with staggered (sticky) ends that will be produced, assuming digestion is fullysuccessful.11 of 14rate during the 20 min incubation period. Centrifuge for 5 s if theECOR1 digestsPUC-4KpBC4KpBC-SKPUC-18

Using restriction endonucleases, a type of enzyme, restriction digestion, also known as restriction…

Answered: biotechnology lab class : 1. The…

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...