cloning protocol, Sally Subcloner wants to clone a fragment into pUC19 (Map above

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)

Write Your Message!

Name

Email id

Message

Verfication Code

User Account

All Pages

cloning protocol, Biology

Related Discussions:- cloning protocol

Animal phylum that contains creatures with analogous organs, Q. Most insect...

.amphibia of frog, functions of liver in frog?

What are gametes, Q. What are gametes? Gametes are cells specialized in...

Anomalies related to oral cavity, Anomalies Related to Oral Cavity: Un...

Points, What is the classification of protozoa with examples

What is the life cycle of a tapeworm, Q. What is the life cycle of a tapewo...

Mosaic and regulative eggs, Mosaic and Regulative Eggs Eggs as well a...

Skin., what are the main functions of basal (Malpighian)layer?

Genetically modified foods-maize, Normal 0 false false fals...

Binomial nomenclature, for assignment

Write Your Message!

Featured Services

Online Tutoring

Project Development

Exam Preparation

Course Help

Assignment Help

Popular Subjects

Assured A++ Grade

Academics

Major Subjects

Majors

Get In Touch

TERMS & POLICIES

HELP & SUPPORT