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

Explain about the microbiological study of water, Explain about the Microbi...

Explain about the Microbiological Study of Water? Water is the basic need of life. River, streams and underground water is the prime source for water. However, disposal of indu

Why water is a essential nutrient for life, Why Water is a essential nutrie...

Why Water is a essential nutrient for life? In this unit, we learnt that water in spite of being ignored, is an essential nutrient required for life. Though the content of tota

Describe shunts, Q. Describe Shunts? Detection, localization and quanti...

Q. Describe Shunts? Detection, localization and quantification of intracardiac shunts are one of the most important exercises in cardiac catheterization. In most cases a prelim

Absorption of amino acids and peptides, Absorption of amino acids and  pep...

Absorption of amino acids and  peptides Generally,  the  dietary proteins are almost comp!etely  digested to their constituent amino acids and these are rapidly absorbed  from

Use of nitrates and nitrites in microorganisms, Q. Explain the Use of nitra...

Q. Explain the Use of nitrates and nitrites in microorganisms? The use of nitrates and nitrites has been widely used in the curing of meat. The reduction of nitrates by bacteri

What is a biome, What is a biome? A biome is the prevailing ecosystem c...

What is a biome? A biome is the prevailing ecosystem constituted by similar abiotic and biotic factors present in one or more regions of the planet.

Explain clostriduim botulinum, Q. Explain Clostriduim botulinum? Clostr...

Q. Explain Clostriduim botulinum? Clostriduim botulinum is widely distributed in soil and marine sediments throughout the. world. It is also found in the intestinal tract of a

Gastrulation, significance of gastrulation

significance of gastrulation

What is arsenite inhibitor, What is Arsenite  inhibitor Arsenite inhib...

What is Arsenite  inhibitor Arsenite inhibits synthesis of ATP,  in the conversion of 1,3-bisphosphoglycerate  to 3- phosphoglycerate, by  causing uncoupling of oxidation and

Blood group, when the father and the mother of a newborn baby possesses blo...

when the father and the mother of a newborn baby possesses blood group ''O'' +ve,is it possible that the baby can have a blood group ''A'' +ve or ''B''+ve ? Conventionally it shoul

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