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 subphylum vertebrata explain, What is Subphylum Vertebrata explain?...

What is Subphylum Vertebrata explain? This large group of animals takes its name from its distinguishing feature: the vertebral column, or the backbone. The backbone made of se

What is cerebral hemorrhage, What is Cerebral Hemorrhage Cerebral hemor...

What is Cerebral Hemorrhage Cerebral hemorrhage is a massive bleeding into the substance of the brain. The most frequent cause is high blood pressure, or hypertension. Other ca

Write at least two possible explanations, Suppose that the type of drink di...

Suppose that the type of drink did not affect which can floated or sank. May be the cans themselves were different in some way. May be something besides soda got into one of the ca

Define some essential facts about the fats, Define some Essential facts abo...

Define some Essential facts about the Fats? 1) Fats are essential in diets to facilitate satiety, high-energy intakes, and absorption of fat-soluble vitamins and provide essent

Explain activators, Activators Activity  of many  enzymes is  influence...

Activators Activity  of many  enzymes is  influenced by  certain ions called as activators. Large number of enzymes such as hexokinase that require ATF are also in need of diva

Swine influenza, Swine influenza Swine influenza, a highly contagious ...

Swine influenza Swine influenza, a highly contagious disease affecting the respiratory tract of pigs, is c a use d  b y a  vir us b e lo nging t o the ge nus I nflu e nz a vir

Explain the marasmus - protein energy malnutrition, Explain the Marasmus - ...

Explain the Marasmus - protein energy malnutrition? Marasmus, the other end of the same spectrum as kwashiorkor, is common in children below the age of 2 years. The characteris

State the jackson coleman and fischer theory, Jackson Coleman and Fischer T...

Jackson Coleman and Fischer Theory Jackson Coleman Theory: During accommodation the ciliary muscle contraction causes an anterior thrust of the vitreous. This vitreous thrust p

Flatworms, What are the adaptions flatworms neeed in order to live in their...

What are the adaptions flatworms neeed in order to live in their environment?

Explain pericardial effusion, Q.Explain Pericardial Effusion? The confi...

Q.Explain Pericardial Effusion? The configuration of the heart in pericardial effusion depends on the volume of fluid and its distribution. It may have a globular or non-speci

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