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

Cardiac cycle, The term cardiac cycle means one pumping cycle which consist...

The term cardiac cycle means one pumping cycle which consists of contraction (systole) and relaxation (diastole) of both the atria and both ventricles. Both the atria contract at t

Coordination of digestion, Coordination of Digestion You have learnt ...

Coordination of Digestion You have learnt that digestion is a process in which large food molecules arc broken down step by step into their constituents. In primitive metazoa

Classification of invertebrates, classes of phylum porifera

classes of phylum porifera

Anaemia, A n a e m i a It is defined as decrease in the amount of...

A n a e m i a It is defined as decrease in the amount of haemoglobin (Hb) per unit of blood. This may or may not be accompanied by a reduction in the red blood cells (RBC

What is echocardiography, Q. What is Echocardiography? Ans. Echoca...

Q. What is Echocardiography? Ans. Echocardiography has become an established and powerful tool for diagnosing presence of CAD and defining its consequence. It can help in

What do you understand by coelenteron, What do you understand by Coelentero...

What do you understand by Coelenteron? The name given to internal cavity of the cnidarians. This is an incomplete gut with just one opening, the mouth. Food to be digested and

Osmosis, If you have water, 10% sucrose, and 40% sucrose solution. Which so...

If you have water, 10% sucrose, and 40% sucrose solution. Which solutions were isotonic to each other?

Undescended testes (cryptorchidism), Undescended Testes (Cryptorchidism) ...

Undescended Testes (Cryptorchidism) Undescended testes is a ccmmon disorder. It may be unilateral and may be classified as ectopic cryptorchidism, when the testes are normal

Medical therapy for aortic stenosis, Q. Medical Therapy for aortic stenosis...

Q. Medical Therapy for aortic stenosis? Definitive therapy for aortic stenosis is surgical and medical therapy is only palliative. Infective endocarditis and rheumatic fever p

Explain about the magnesium - macro minerals, Explain about the Magnesium -...

Explain about the Magnesium - macro minerals? Magnesium (Mg) ranks fourth in overall abundance in body among the cations. It is also the least abundant among macro minerals, th

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