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

Role of chemical factors in controlling apical dominance, Role of Chemical ...

Role of Chemical Factors in Controlling Apical Dominance There were indications about the existence of plant hormones in the last part of 19th century. The plant hormones had

Show the natural selection of taxonomist, Q. Show the Natural selection of ...

Q. Show the Natural selection of taxonomist? Natural selection associated with successful reproduction maintains a basic similarity of the reproductive feature of flowers, frui

What is working of mouth in human body, What is Working of Mouth in human b...

What is Working of Mouth in human body? Food enters the digestive system through the mouth, where it is processed by the teeth and mixed with saliva, prior to being swallowed.

Cell, Ask question #Minimum cell theory 100 words accepted#

Ask question #Minimum cell theory 100 words accepted#

Explain natural history of mitral stenosis, Q. Explain Natural History of M...

Q. Explain Natural History of Mitral Stenosis ? Untreated, mitral stenosis is a serious disease with observed 10-year mortality of 70-80 per cent in sy

Explain about microwave irradiation, Q. Explain about Microwave irradiation...

Q. Explain about Microwave irradiation? Most micro-organisms are destroyed by the action of microwave of a frequency of about 2450 MHz and a wavelength of 12.24 cm. The water c

Explain gellan gum, Explain Gellan Gum Gellan is an exopolysaccharide c...

Explain Gellan Gum Gellan is an exopolysaccharide consisting of  β-D-glucose,  β-D-glucuronic acid,  α-L-rhamnose with acetate and glycerate groups, produced by the bacterium S

What do you mean by amphibians of the plant world, Q. Why can the bryophyte...

Q. Why can the bryophytes be considered the "amphibians of the plant world"? Like adult amphibians, the bryophytes live in the terrestrial environment but they depend on water

Tissue, What important changes will you find in the tissue of the fat perso...

What important changes will you find in the tissue of the fat person from those of a lean and thin person

What is consanguineal marriage, What is consanguineal marriage? Why is the ...

What is consanguineal marriage? Why is the appearing of genetic disease more probable in the offspring of a consanguineal marriage? Consanguineal marriage is the marriage among

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