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

How different are cnidarians from poriferans, Concerning tissue complexity ...

Concerning tissue complexity how different are cnidarians from poriferans? Cnidarians have true tissue differentiation, they present separate organized tissues in the body. Por

Chemoreceptors, Chemoreceptors These are receptors included in percept...

Chemoreceptors These are receptors included in perception of chemical stimuli. You will see that there are three kinds of Chemoreceptors among metazoans:  i) Those concerne

Transcription, function of topoisomerace?

function of topoisomerace?

What is recombination frequency?, What is recombination frequency? The ...

What is recombination frequency? The Recombination frequency, or crossing over rate, is the percentage of recombinant gametes made by crossing over (in relation to the number o

What are meninges and cerebrospinal fluid, What are meninges and cerebrospi...

What are meninges and cerebrospinal fluid? Meninges are the membranes that enclose and protect the central nervous system (CNS). Cerebrospinal fluid is the fluid that separates

What is phylum nematoda - roundworms, What is Phylum Nematoda - Roundworms?...

What is Phylum Nematoda - Roundworms? Nematodes are commonly known as the roundworms. They have a pseudocoelom type of body cavity, and they have a digestive tract that opens t

What are allosteric enzymes, Allosteric enzymes are those that have more ac...

Allosteric enzymes are those that have more activation center and to which other substances known as allosteric regulators bind. Allosteric regulators can be allosteric inhibito

Charecteristics of invertebrates, What is general characteristics of invert...

What is general characteristics of invertebrates

What are extraembryonic membranes, Q. What are extraembryonic membranes? ...

Q. What are extraembryonic membranes? Extraembryonic membranes are membranous structures that appear in parallel with the embryo and play vital roles in the embryonic developme

Yersinia enterocolitica, Yersinia enterocolitica The genus Yersinia con...

Yersinia enterocolitica The genus Yersinia contains of 3 human pathogenic species, Y. enterocolitica, Y. pseudotuberculosis and Y. pestis, among which, Y. enterocolitica finds

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