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 glands, What is Glands? The central nervous system controls muc...

What is Glands? The central nervous system controls much of the endocrine system through the hypothalamus. The hypothalamus directs many of the body's functions through the "ma

The first carbon having 2 h atoms, Given a polymer backbone of H-C-C with t...

Given a polymer backbone of H-C-C with the first carbon having 2 H atoms attached and the 2nd carbon with 3 atoms attached, a bond with a water molecule attaches to the left H atom

Explain the s1m filament theory of muscle contraction, Explain the s1M fila...

Explain the s1M filament theory of muscle contraction. What is special of "FlavrSavr" variety of tomato? Why is it preferred to its normal native variety? Illustrate a label

Paleomon., coxa of paleomon contain specialised organ called

coxa of paleomon contain specialised organ called

How is the early diagnosis of genetic diseases usually done, How is the ear...

How is the early diagnosis of genetic diseases usually done? Genetic disease might be diagnosed in the prenatal period by karyotype analysis, in case of aneuploidies, or by DNA

Pteridophytes, What are the affinities of pteridophytes with gymnosperms?

What are the affinities of pteridophytes with gymnosperms?

Gifted species - biological nitrogen-fixation, Gifted Species - Biological ...

Gifted Species - Biological Nitrogen-Fixation Biological nitrogen fixation remains mainly confined to a few distinct nutritional types of prokaryotes, some of which are free-l

Explain the phylum molluska - mollusks, Explain the Phylum Molluska - Mollu...

Explain the Phylum Molluska - Mollusks? You are probably very familiar with members of this phylum. The mollusks include the octopuses, squids, cuttlefish, scallops, oysters, c

What are prions? define concerns of food safety, Normal 0 false...

Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4

Abrasive action of water and wind, Abrasive action of water, ice and wind ...

Abrasive action of water, ice and wind Water is an important mechanical weathering agent both in liquid as well as in the frozen state.  Rain water carrying sediments, ice glac

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