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

Determine some beneficial effects of phytates, Determine some Beneficial ef...

Determine some Beneficial effects of phytates? Some of the health promotive aspects of phytates include: 1) Phytic acid is a known antioxidant. Colonic bacteria produce oxyg

Functions of cell wall, FUNCTIONS OF CELL WALL 1.       Provides shape ...

FUNCTIONS OF CELL WALL 1.       Provides shape to plant cell rigidity to cells. 2.       Functions as a barrier to entry of pathogens into the cells. 3.       Provides pr

Regeneration and its examples, R E GENER A TIO N - Natural ability...

R E GENER A TIO N - Natural ability of living being to replace worn out parts, to repair damaged parts, to regrow cost off organs & to reform entire body from a small piec

Excretory system of animals and human urinary system, steps in urine format...

steps in urine formation and the organs/tissues involve in such process?

A monohybrid cross, A monohybrid cross: A.Determines the genetic makeup of ...

A monohybrid cross: A.Determines the genetic makeup of an organism B.always involves homozygous alleles. C.always involves organisms that are heterozygous at all loci. D.Always inv

Pathogens, Give a brief overview of "HIV & Streptococcus Pyogenes" includin...

Give a brief overview of "HIV & Streptococcus Pyogenes" including signs and symptoms. Who is typically affected by this infection?(Children? Immunocompromised patients?)? What is t

Absorbing surface of the small intestine is increased, State three ways in ...

State three ways in which the absorbing surface of the small intestine is increased.   The absorbing surface of the small intestine is increased by:- (a) being very long

What are food hydrocolloids, What are food hydrocolloids? Food hydroco...

What are food hydrocolloids? Food hydrocolloids are hydrophilic polymers of vegetable, animal, microbial or synthetic origin that generally contain many hydroxyl groups and ma

Social determinants of health - food, Social Determinants of Health - Food ...

Social Determinants of Health - Food The twin paradox of food i.e., research showing excessive intake can lead to a variety of diseases whilst at the same time food poverty be

Biotic potential of a bacterial population, Q. How different is the growth ...

Q. How different is the growth according to the biotic potential of a viral population from the growth according to the biotic potential of a bacterial population? The growth c

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