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

Define intentional adulteration - types of adulteration, Define Intentional...

Define Intentional Adulteration - Types of Adulteration? In intentional adulteration, the substance is added, removed or substitute knowingly by the adulterator for the purpose

Neck examination of new born, Neck Inspect and palpate the neck for an...

Neck Inspect and palpate the neck for any mass, check if neck is short, and webbed which is indicative of Turner's syndrome. Palpate posteriorly along cervical spine; later

Excretion and the kidneys, short notes on cholesterol.

short notes on cholesterol.

Orthopedics, 1. List alternative therapies for musculoskeletal problems. ...

1. List alternative therapies for musculoskeletal problems. 2. What is a xyrospasm? What is the Greek derivative of the word? 3. Name and describe the three types of varus con

Define the classification of proteins, Define the Classification of Protein...

Define the Classification of Proteins? Proteins vary widely in. their properties. We suggest you look up this unit once again now as the information about proteins and their pr

What are main successful vaccines, Main successful vaccines are live attenu...

Main successful vaccines are live attenuated and heat killed vaccines.

Which are the most important inorganic molecular substances, Q. Which are t...

Q. Which are the most important inorganic molecular substances for living beings? Ans. The most important inorganic substances for living beings are mineral salts, water,

Effect of a deletion in the operator of the lactose operon, Compare and con...

Compare and contrast the effect of a deletion in the operator of the lactose operon with one in the operator of the histidine operon.

What is rhizopus - fungi, What is Rhizopus - Fungi? Rhizopus is a commo...

What is Rhizopus - Fungi? Rhizopus is a common laboratory contaminant. It is a spoiling mould and found frequently on the surface of bread, fruits and vegetables. It can grow a

Autoradiography, Autoradiography Autoradiography is a modification of t...

Autoradiography Autoradiography is a modification of the radioactive tracer technique. In this technique, the radioactivity is detected by a thin film of photographic emulsion

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