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

Glucocorticoid response element, GRE  stands for the Glucocorticoid Respons...

GRE  stands for the Glucocorticoid Response Element: The binding site in a promoter to which the activated glucocorticoid receptor can be bind. The glucocorticoid receptor is signi

Methods of curing meat, M e t h ods of curing Curing ingredients ar...

M e t h ods of curing Curing ingredients are applied in dry form or as a solution of curing ingredients in water (pickle or brine). The combination cure is applied in the i

Explain continuous murmurs and their characterstic, Explain Continuous Murm...

Explain Continuous Murmurs and their characterstic in details? These murmurs are continuous throughout systole and diastole. It is due to continuous blood flow in the same dire

Cellular specific immune response, Q. What is the difference between cellul...

Q. What is the difference between cellular specific immune response and humoral specific immune response? Humoral specific immune response is the defense system by means of, an

How are roots affected by water, How are roots affected by water? Grow ...

How are roots affected by water? Grow some seedlings in single end of a glass dish or pan. When they are about 5 cm tall start watering them on one side only and a little dista

Canine distemper, Canine distemper Canine distemper, a highly contagio...

Canine distemper Canine distemper, a highly contagious disease of dogs, is caused primarily by air- borne virus which belongs to the genus Morbillivirus in family Paramyxoviri

Explain chemical structure of saponins, Explain Chemical Structure of sapon...

Explain Chemical Structure of saponins? Let us briefly understand their chemical nature. Their structures are characterized by the presence of a steroid or triterpene group, i.

Define the basic concepts behind the spectral techniques, Define the Basic ...

Define the Basic Concepts behind the Spectral Techniques? Energy is transmitted via electromagnetic waves that are characterized by their frequency and wavelength. The physical

Syngamy, Syngamy The pollen tube grows to a very limited extent in the...

Syngamy The pollen tube grows to a very limited extent in the synergid. It releases the contents either through a terminal or a subterminal pore. The contents include the two

What are the main water properties, What are the main water properties that...

What are the main water properties that make water special for life? The water properties that make water biologically significant are molecular polarity, thermal stability (el

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