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

Bomb calorimeter, Normal 0 false false false EN-IN X-...

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

Energy, what are the types of pyramids studied

what are the types of pyramids studied

Extremes of heat and cold, Extremes of Heat and Cold Deserts are region...

Extremes of Heat and Cold Deserts are regions of aridity with rainfall of less than 20 cm per year and the soil, though L, fertile, is too porous to retain any water. In summer

Procedures for diagnosis - nucleic acid hybridization, Nucleic acid hybridi...

Nucleic acid hybridization: This is a process by which a suitably labeled DNA or RNA from a known infectious agent is used to probe its complementary sequence unique to t

What are cotyledons, What are cotyledons? Cotyledons or else seed leave...

What are cotyledons? Cotyledons or else seed leaves are structures formed by the embryo of angiosperms to absorb nutrients from the endosperm and to store and transfer these nu

Explain faecal coliforms - microbiological study of water, Explain the Faec...

Explain the Faecal Coliforms - Microbiological Study of Water? The faecal coliforms include a wide range of bacteria, many of which are not primarily the intestinal bacteria. T

Orifices of heart, Atrio-ventricular Orifices The right and left atrio-v...

Atrio-ventricular Orifices The right and left atrio-ventricular orifices are oval apertures. The openings lie in a plane that is almost vertical with a slight downward inclinati

Explain the eligibility of cardiac rehabilitation, Explain the eligibility ...

Explain the eligibility of Cardiac rehabilitation ? Cardiac rehabilitation services are prescribed for patients who: 1) Have had a myocardial infarction. 2) Have had coronary b

Phylum annelida, Classification

Classification

What is the definition of ecology explain, What is the Definition of Ecolog...

What is the Definition of Ecology explain? Scientists use the term ecology to refer to the study of the relationships between organisms, and the relationships between organism

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