?l?smid R?stri?ti?n ????ing

Plasmid Restriction Mapping Answerthe Pre-Iaboratory Questions 1) An example of aplasmid map is shown below. What is a plasmid map and why is it useful. Figure

3.1 Plasmid map of pSH471 2) Enzymatic restriction of a plasmid can be a single digest, a dpubIeddigest?or a triple digest. What doesthis mean and what are how

many DNAfragments are expected if each ofthe enzyme only cutsthe p asmi once. 3) Why isthe use of restriction enzymes an important tool for plasmid mapping? 4)

Describe the process of how you might map a plasmid using restriction enzymes. 5) Have you read the Risk Management documentforthis practical and are you

readyto comply with the recommended risk control measures? Part 2: Discussed and completed the following Questions: 1) A plasmid was cut using two restriction

enzymes (EcoRI and Hindlll), both individually and together. The samples were then run on an agarose gel, and the size of each fragment was recorded (see below).

Use this information to answerthe following2: Sample Size of Band/s Observed (bp) EcoRI 30, 15, 5 Hindlll 50 EcoRI and Hindlll 20,15,10,5 Find a plasmid map of

pBR322 and work outwhat would be the expected fragments sizes when this plasmid is cut by the following restriction enzymes: d) EcoRI e) Pv ull/EcoRI f)

EcoRI/SaII E) Sall/Pv ull EcoRI/Sall/Pvull SHOWALL YOUR WORKING IN CALCULATING THE FRAGMENT SIZES. 2) Acircular bacterial plasmid (pBP1) contains in the

tetracycline-resistance gene (tetR)3. Human genomic DNA has been digested and inserted into tetR ofthe plasmid. This particular plasmid clone (number 15)

contains the cloned gene.You perform restriction digests with Hindlll and EcoRV The gel results are shown above,with the control being plasmid with no insert. Band

sizes are given in kilobases (kb). You can assume that all bands are from linear molecules. Answerthe questions below. a) Why might it be useful to have a

restriction site in the middle of an antibiotic resistance gene of a cloning vector such as a plasmid? b) How manyfragments are produced ifthe pBP1 plasmid is cut

with an enzyme that cutstwice? How many fragvments would be produced ifthis was a linear piece of DNA? c) hat does the Hindlll site tell you? Why? d) What three

things does the EcoRV site tell you? Why? e) Drawtwo simple maps ofthis plasmid – one with and one withoutthe insert, marking the possible restriction sites of

Hindlll and EcoRV. Also mark the sizes of regions in between. 3) Construct a restriction map of a circular DNA p asmid, using the following data. Your map should

indicate the relative positions ofthe restriction sites along with distances between restriction sites: DNA Sizes of Fragments (bp) uncut DNA 7950 DNA cut with Bglll

7950 DNA cut with EcoRI 7950 DNA cut with Hpal 7950 DNA cut with Bglll + EcoRI 5416, 2534 DNA cut with Bglll + Hpal 6632,1318 DNA cut with EcoRI + Hpal

4098, 3852 4) Shown below is a DNAfragmentwhich contains a newly discovered gene, Gene I. You wish to subclone Gene I into a vector in order to study it. The

restriction enzyme EcoRI is chosen to digest the target DNA and the vector DNA, and the EcoRI fragment containing the open rea ing frame of Gene I is igated

into the EcoRI site ofthe vector. Answerthe questions below. a) How many orientations, relative to promoterX in the vector, could the insert fragment be in? (Draw

a map ofvector +insert for each possible orientation in the space below) b) Which single restriction enzyme(s) would you use to determine the orientation, relative

to promoterX? Indicate the fragment that would be released belowthe map(s) you have drawn and explain why you selected these particular enzymes and not

others. C) Under what circumstances would it be importantthat the insert was in the orientation such that the direction ofthe open reading frame in Gene I isthe

same as the direction oftranscription from promoterX? d) If you wished to ensure that the insertwas cloned in this orientation and thatthe entire gene was

inserted,which two enzymes could be used instead to subclone the fragment containing Gene I into the vector? Explain why you selected these and not others.

Conclusion Think carefully about the practical/workshop and list what information you have learnt from it. Laboratory report requirements: Thzre is no formal report

to write for this practical. What you need to o: Answers to pre-Iaboratory questions in details Discussthe practical questions with evidences Answers all the

problems scientifically (thisthe most important part) One paragraph Conclusion. ‘please see the attached PDF file for practical informaion

Explain agency rulemaking procedures; compare and contrast the different types of rulemaking procedures.
Describe the process of judicial review and discuss the scope of review and the standards of review

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