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

Genetics , Genetics Genetics is the study of heredity. It is an ancien...

Genetics Genetics is the study of heredity. It is an ancient discipline. At least 4000 years ago, in Sumeria, Egypt and other parts of the world, farmers recognized that they

Rheumatic fever and rheumatic heart diseases, Rheumatic Fever and Rheumatic...

Rheumatic Fever and Rheumatic Heart Diseases Rheumatic fever is an inflammatory disease of the heart potentially involving all layers of the heart. The resulting damage to t

Neuston - aquatic ecosystem, Neuston - Aquatic Ecosystem These are una...

Neuston - Aquatic Ecosystem These are unattached organisms which live at the air-water interface such as floating plants and several types of animals. Some spend most of their

What function present in placental mammalian embryos, Q. Which is the extra...

Q. Which is the extraembryonic membrane whose function is to store nitrogen wastes of the embryo? Is this function present in placental mammalian embryos? The allantois is the

Under nourished persons - nutritional intervention using mna, Define Under ...

Define Under nourished persons - nutritional intervention using MNA? An MNA score below 17 indicates that the patient is suffering from under nutrition.  Thus in this situatio

Define classification of carbohydrates - polysaccharides, Define classifica...

Define classification of carbohydrates - Polysaccharides? This classification is based on the number of monosaccharide units they contain, Polysaccharides are those polymers of

Chromosomes, how many arrs are present in mataphasic telocentric chromosome...

how many arrs are present in mataphasic telocentric chromosomes

Explain xanthan gum, Explain Xanthan Gum Xanthan gum is a microbial pol...

Explain Xanthan Gum Xanthan gum is a microbial polymer prepared commercially in pure culture fermentation from Xanthomonas campestris. This polysaccharide was given the generic

#title.circulatory system, #question.what is the mechanism of circulatory s...

#question.what is the mechanism of circulatory system .

Determine the high blood plasma levels of insulin, A patient has total dest...

A patient has total destruction of all of the beta-islet cells of the pancreas.  Prior to receiving treatment, this patient will have A. high blood plasma levels of glucose.

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