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

Define the dietary guidelines, Define the Dietary Guidelines? Dietary G...

Define the Dietary Guidelines? Dietary Guidelines are becoming increasingly powerful tools to help the general public to appreciate the role of diet in prevention of degenerati

Nutritional management of diabetes mellitus, Q. Nutritional management of d...

Q. Nutritional management of diabetes mellitus? we will learn about the nutritional management of diabetes mellitus which is an important metabolic disorder of public health si

Fats requirement in pectic ulcer, Q. Fats requirement in pectic ulcer? ...

Q. Fats requirement in pectic ulcer? Fats: These delay the gastric emptying. The products of fat digestion in the small intestine stimulate entrogastrone, which inhibits gastri

What is isozymes, ISOZYMES : an enzyme having different molecular forms bu...

ISOZYMES : an enzyme having different molecular forms but catalyzing the same reaction.

Pressure gradient, Pressure Gradient You know that air pressure is the ...

Pressure Gradient You know that air pressure is the weight of the atmosphere over a unit area of the earth's surface. The average air pressure at sea level is approximately 1 k

#title.modes of nutrition, what are the modes of nutrition in different ani...

what are the modes of nutrition in different animals

Phylum Coelenterata, What is the adaptative radiatoin of coelenterates

What is the adaptative radiatoin of coelenterates

Modes of respiration, Modes of Respiration Many small organisms obtain...

Modes of Respiration Many small organisms obtain oxygen by diffusion through their body surfaces. They do not have any specialised respiratory organs nor do they have blood ci

Define the concept of ventilation, Which of the following are true for vent...

Which of the following are true for ventilation? A. The problems with ventilation induced by injection of curare occur because of the drug's direct action on nicotinic ACh Rec

Determine food sources for zinc, Determine Food Sources of Zinc? Zinc i...

Determine Food Sources of Zinc? Zinc is normally associated with the protein and/or nucleic acid fraction of foods. Thus, foods high in proteins are good sources of zinc. Lean

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