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

Water loss - plant water relation, Water Loss - Plant Water Relation ...

Water Loss - Plant Water Relation Plants lose about 98% of water to the atmosphere by transpiration. Often water loss by transpiration exceeds gain by absorption and results

What is polypropylenes, Q. What is Polypropylenes? Polypropylenes (PP) ...

Q. What is Polypropylenes? Polypropylenes (PP) (cast PP, Bi-axially oriented PP (BOPP), pearlized PP): Polypropylene is often termed a breathable film. This film is not a

State the process of rna editing, Which of the following is a true statemen...

Which of the following is a true statement regarding the process of RNA editing? A. RNA editing changes the sequence of the mRNA message thereby permitting for enhance in prote

Types of osmotic exchanges, Types of Osmotic Exchanges The osmotic exc...

Types of Osmotic Exchanges The osmotic exchanges that take place between an animal and its environment are of two different types: Obligatory exchanges and Regulated

Similar organs and similar general shape be explained, How can the fact tha...

How can the fact that fishes and dolphins have similar organs and similar general shape be explained? Dolphins and Fishes have similar organs and shape because although they ha

Situation in which the mistakes holds in dna replication, Q. Mistakes may h...

Q. Mistakes may happen during each copying process. The same is true for the DNA replication. Are there correction systems in cells that try to mend those mistakes? Under which sit

Illustrate artificial active immunization, Q. What are artificial active im...

Q. What are artificial active immunization and natural active immunization? Natural active immunization is that in which a previous natural infection induces the primary immune

Active or moderately active lifestyles - physical activity, Define Active o...

Define Active or moderately active Lifestyles - physical activity? These people have occupations that p-e not strenuous in terms of energy demands, but involve more energy expe

Respiration in animals, Respiration Robert Boyel and Robert Hook fir...

Respiration Robert Boyel and Robert Hook first of all explained the meaning of respiration. Main aim of respiration is to liberate energy in the form of A.T.P. Oxidati

Define the beer''s law - nutritional biochemistry, Define the Beer's Law -...

Define the Beer's Law - Nutritional  Biochemistry? Beer's law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely

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