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

What is the autophagic intracellular digestion, Q. What is the autophagic i...

Q. What is the autophagic intracellular digestion and why is this type of intracellular digestion intensified in an organism undergoing starvation? Autophagic intracellular dig

Determine the prosthetic planning, Determine the prosthetic planning Co...

Determine the prosthetic planning Complete prosthetic planning has to be done and the implants planned accordingly. Thereafter the surgical phase should be carried out. Once th

Elaborate the term - gill slits, Elaborate the term - Gill slits. Later...

Elaborate the term - Gill slits. Lateral opening in the wall of pharynx which allows water to enter into mouth and exit through the pharynx. This is one of the ancestral charac

How does the intensity of facilitated diffusion differ, Q. How does the int...

Q. How does the intensity of facilitated diffusion differ in relation to the concentration of the moved substance? What is limiting factor? Like simple diffusion facilitated di

What are the results of chrchronic pulmonary thromboembolism, What are the ...

What are the Results of Chronic Pulmonary Thromboembolism? Results :  The reported mortality is between 5 and 24 per cent. On follow-up, the six-year survival in the best ce

Fensom and spanner electroosmotic flow hypothesis, Fensom and Spanner Elect...

Fensom and Spanner Electroosmotic Flow Hypothesis In electroosmosis the ions flow across a membrane in response to electrical gradient. Ions pull along water and other content

Pattern of growth, P A TTERN OF GROWTH - 1 .       ISOMETRIC - ...

P A TTERN OF GROWTH - 1 .       ISOMETRIC - In it an organ grows at the same rate as the rest of body. External form of body is not changed. eg. fish, locust. 2 .

Cell cycle, what is the process of cell cycle?

what is the process of cell cycle?

Genetics, what is cell

what is cell

Epizootic lymphangitis, Epizootic lymphangitis Epizootic lymphangitis ...

Epizootic lymphangitis Epizootic lymphangitis (pseudo-glanders) is a chronic, insidious and highly contagious fungal disease of equines particularly horse and very rarely of c

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: +1-415-670-9521

Phone: +1-415-670-9521

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

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