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

Spreading mechanisms of the disease, Talk about briefly three  of the commo...

Talk about briefly three  of the common enteric diseases caused by inadequate clean water supply and insufficient sanitation facilities. Your talk should contain the source and spr

Mounting boxes for insect collections, Mounting boxes for insect collection...

Mounting boxes for insect collections Wood or cardboard cigar boxes make very useful and suitable housings for insect collections. After the insect has been separate from the s

DNA copying, estion..

estion..

Formation of chiasmata, Chiasmata formation takes place where crossing over...

Chiasmata formation takes place where crossing over occurs. Here Chromatid segments are exchanged which contributes to genetic variability. The 46 homologous chromosomes arrange at

Calcium channel blockers, Calcium antagonists are not recommended for the t...

Calcium antagonists are not recommended for the treatment of CHF because of their negative inotropic effects. However, second-generation dihydropyridine-type calcium antagonists su

What is biomolecules?, A biomolecule is any molecule that is produced by a ...

A biomolecule is any molecule that is produced by a living organism, including large macromolecules like proteins, polysaccharides, lipids, and nucleic acids, as well as small mole

Korarchaeotes, what is the effect that korarchaeotes have on humans?

what is the effect that korarchaeotes have on humans?

Define locust bean gum, Locust Bean Gum Locust bean gum (also called Ca...

Locust Bean Gum Locust bean gum (also called Carob bean gum) is extracted from the seed (kernels) of the carob tree (Ceratonia siliqua). Structurally, locust bean gum is compos

Differentiate between neuropsychologists and neurologists, Differentiate be...

Differentiate between neuropsychologists and neurologists Cooperation among neuroraudiologists, neurologists, and neuropsychologists has already led to the accomplishment of se

Respiratory gases -respiration, Respiratory Gases -Respiration Physio...

Respiratory Gases -Respiration Physiologically the most important gases are oxygen, carbon dioxide and nitrogen. Molecular oxygen comprises 21% of the atmosphere; carbon diox

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