cloning protocol, Biology

Assignment Help:
Sally Subcloner wants to clone a fragment into pUC19 (Map above – From New England Biolabs). Her fragment was a protein coding gene of 2.7 kb, and had XhoI sites on either end (and no other XhoI sites any where else) and she agarose gel purified this fragment to ensure it was pure. As no XhoI sites existed in the pUC19 plasmid, she decided to clone it into the Sal1 site, as Sal1 and Xho1 have identical overhangs of 5’-TCGA-3’ and therefore she believes that they can be ligated to one another.

As she was using blue/white screening she did not bother with Alkaline Phosphate treatment and ligated the XhoI fragment directly with SalI digested pUC19. After transformation into E. coli (strain AG1 using heat shock treatment) she plated cells onto media with Ampicillin selection (100 mg/mL) and obtained over 300 ampicillin resistant colonies. However none of these were white colonies, only blue, which she interpreted as obtaining no plasmids with the fragment she was aiming to subclone.

(a) Was her cloning approach valid and what was the reason that she could not get white colonies from this protocol? If she modifies the protocol to address this question, what additional advice can you give Sally Subcloner to increase her chances of obtaining white colonies. (10 Marks)

(b) After taking this advice and obtaining white colonies, she purified the plasmid from five different white colonies digested them with Xho1 and ran them on a gel (see below). However there were three bands in each lane, with the major band running at approximately 4.5 kb, instead of 2.7 kb that she expected her XhoI fragment to be. Could these plasmids still be the right recombinant and what simple experiment could she perform to determine this? (Hint what control should have been included in the gel?) (10 Marks)

Related Discussions:- cloning protocol

New Technology, What is the best microscope to get a detailed view of the p...

What is the best microscope to get a detailed view of the parts inside of a preserved plant cell

Dissection, a 6th grade student friendly definition?

a 6th grade student friendly definition?

Define the properties of fibre, Define the Properties of Fibre? The str...

Define the Properties of Fibre? The structural make up of fibre influences its properties which in turn affects the physiologic and metabolic roles. This is well-depicted in th

Pseudocoelom - metazoa, Pseudocoelom - Metazoa The platyhelminths whic...

Pseudocoelom - Metazoa The platyhelminths which do not have a body cavity surrounding the gut, have a solid type of body constitution. The mesoderm completely fills the space

Differ from our current use of retroviruses, In what way may we be able to ...

In what way may we be able to take advantage of retrotransposons in human gene therapy? How would this differ from our current use of retroviruses?

What is adp phosphorylation, What is ADP phosphorylation? What respectively...

What is ADP phosphorylation? What respectively are photophosphorylation and oxidative phosphorylation? ADP phosphorylation is the addition of one inorganic phosphate in the mol

Explain lyases, Explain lyases These enzymes (code EC 4) cleave C-C,  C...

Explain lyases These enzymes (code EC 4) cleave C-C,  C-O, C-N  and other bonds by elimination, forming double bonds or conversely adding groups  to double bonds. Common names

Name the food yersinia enterolytica infection isolated from, Normal 0 ...

Normal 0 false false false EN-IN X-NONE X-NONE MicrosoftInternetExplorer4 Determine the Yersin

Categories of air pollutants, Categories of Air Pollutants From the ab...

Categories of Air Pollutants From the above list you can see that the air pollutants can be broadly classified into the following two categories: Primary Pollutants

How two organisms are interdependent, Give an example of how two organisms ...

Give an example of how two organisms are interdependent. Animals generate carbon dioxide and water, which is required by plants. Accept all reasonable answers.

Write Your Message!

Captcha
Free Assignment Quote

Assured A++ Grade

Get guaranteed satisfaction & time on delivery in every assignment order you paid with us! We ensure premium quality solution document along with free turntin report!

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd