User Account

All Pages

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

Cells are miniature chemical factory, Cells is a Miniature Chemical Factory...

Cells is a Miniature Chemical Factory Due  to its incessant metabolic processes, a living   cell earns the  attribute  of a  miniature chemical factory   metabolites and  free

Explain cidofovir, Cidofovir (Vistide) IV cidofovir given once weekly ...

Cidofovir (Vistide) IV cidofovir given once weekly for 2 weeks and then once every 2 weeks for maintenance therapy can delay progression of CMV retinitis in patients with AIDS

Nutritional management for patient of alzheimer''s disease, Define Feeding ...

Define Feeding and Nutritional Management for patient of alzheimer's disease? Keeping the clinical manifestations of Alzheimer's disease in mind, treatment involves personaliz

Show the regulation of blood pressure, Q. Show the Regulation of blood pres...

Q. Show the Regulation of blood pressure? Nervous System control of BP Maintains primarily an adequate systemic BP, and alters blood distribution in body to meet specific fu

Zoonotic diseases-influenza, Influenza Influenza is an acute infectious di...

Influenza Influenza is an acute infectious disease caused by influenza viruses of genus Orthomyxovirus in family Orthomyxoviridae. The name Influenza is derived from an Italian ph

Define the agarose gel electrophoresis (age), Define the Agarose gel electr...

Define the Agarose gel electrophoresis (AGE)? This technique is used to analyze serum proteins, haemoglobin variants, lactate dehydrogenase isoenzymes, lipoprotein fractions an

How is respiration performed by the larva of amphibians, Q. How is respirat...

Q. How is respiration performed by the larva of amphibians? The larva of amphibians has exclusively branchial respiration this is one of the reasons why it depends on water to

Initiation of aseptic cultures, Initiation of Aseptic Cultures The nut...

Initiation of Aseptic Cultures The nutrient medium (basal medium) used for plant tissue culture would promote luxuriant growth of micro-organisms such as bacteria and fungi. W

Explain the management strategies congenital heart disease, Explain the Man...

Explain the Management Strategies for Adults with Congenital Heart Disease ? The goals for management of congenital heal disease in adults are improving upon the natural histo

How does plasma membrane regulate, How does a plasma membrane regulate move...

How does a plasma membrane regulate movement of molecules into and out of a cell?

Write Your Message!

Captcha
Order Assignment

Featured Services

Order Now

Popular Subjects

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!

Submit Assignment

Academics

Major Subjects

Majors

Get In Touch

whatsapp: +91-977-207-8620

Phone: +91-977-207-8620

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

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