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

What is the requirements of dental implants, Requirements of Dental implant...

Requirements of Dental implants Dental implants require special oral hygiene techniques. The particular technique chosen often depends upon the prosthesis.  A removable prosth

Explain metabolic requierments for types of exercise, Explain Metabolic Req...

Explain Metabolic Requierments For Types Of Exercise ? A relative scale of energy requirements related to heart function necessary for performing various daily activities has b

Carpentier-technique of operation, Carpentier: Edwards or similar annul...

Carpentier: Edwards or similar annuloplasty ring is then sutured into place by interrupted sutures to support the repair and reduce the annulus to a measured amount. The ring m

Angiographic quantification, Angiographic Quantification Gr 1 - minimal...

Angiographic Quantification Gr 1 - minimal systolic jet, clears rapidly Gr 2 - partial RA opacification Gr 3 - Opacification of whole of RA GR4 - Opacification of

In what ways is genomic imprinting similar to x-inactivation, Based on the ...

Based on the simplified two-gene model for eye colour, explain using genotypes how two blue-eyed parents could produce a brown-eyed child. In what ways is genomic imprinting sim

Explain alterations occurring in milk and milk products, Alterations occurr...

Alterations occurring in milk and milk products In the dairy industry, milk is commonly given heat treatment for a wide variety of purposes. Depending on the heating temperatur

Active listening - counselling skills used in diabetes, Q. Active Listening...

Q. Active Listening - counselling skills used in diabetes? Every day we listen to many things, may be not with any intention or paying attention to the words, speech etc. For i

Explain human development, Explain Human Development? Human development...

Explain Human Development? Human development begins with fertilization, and continues throughout life. Fertilization occurs when two sex cells, a sperm cell and an egg cell, jo

Character of phylum annelida, what are the advanced characters of phylum an...

what are the advanced characters of phylum annelida that cannot be found in other phyla?

Write Your Message!

Captcha
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!

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd