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

Discuss in brief about headaches, Discuss in brief about the Headaches ...

Discuss in brief about the Headaches Headache is so common among the general population that rare indeed is the person who has never suffered one. Headache may constitute a neu

Hybridoma, Hybridoma is the clone of plasmacytoma cells which secrete a mo...

Hybridoma is the clone of plasmacytoma cells which secrete a monoclonal antibody; commonly produced by the fusion of peripheral or splenic plasma cells taken from the immunized mo

Explain the anaesthesia and analgesia, Explain the Anaesthesia and Analgesi...

Explain the Anaesthesia and Analgesia Most implant surgery can be carried out under local anaesthesia, although some patients will require sedation or general anaesthesia. The

Important causes of degradation of ecosystem, Important Causes of Degradati...

Important Causes of Degradation of Ecosystem The important causes that have lead to the degradation of the ecosystem. Deforestation Overgrazing Agriculture

Phylum, description of first five phylum

description of first five phylum

How are mammals characterized, Q. Mammal identity card. How are mammals cha...

Q. Mammal identity card. How are mammals characterized according to examples of representing beings, skin, respiration, nitrogen waste, basic morphology, circulation, thermal contr

Animal viruses, Chart of taxonomy of animal viruses

Chart of taxonomy of animal viruses

State the enzymes and recombinant dna technology, Enzymes and recombinant D...

Enzymes and recombinant DNA technology Recombinant DNA technology, also called as genetic engineering makes use of a variety of enzymes, particularly restriction endonucleases

Define influence of gardening on the development of botany, Define Influenc...

Define Influence of Gardening on the Development of Botany? The influence of gardening on the development of botany may be seen in the work of John Gerard (1545-1612). Gerard p

Explain the behavioural change communication, Explain the Behavioural chang...

Explain the Behavioural change communication? One of the reasons for the widespread prevalence of PEM in our country is ignorance due to illiteracy, particularly among the fema

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