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

Enzyme creates double bonds, Suppose you treated butter with a fatty acid d...

Suppose you treated butter with a fatty acid desaturase, an enzyme that removes hydrogen from fatty acids and creates double bonds. Please explain what would happen?

Inverterbrate zoology, economic importance of the phylum protozoa

economic importance of the phylum protozoa

Define sample collection and estimation of blood glucose, Define Sample col...

Define Sample collection and estimation of blood glucose? The aim of blood glucose estimation is to diagnose or to exclude or to monitor the anti-diabetic therapy. For blood gl

Define the streak plate method - pure culture techniques, Define the Streak...

Define the Streak Plate Method - Pure Culture Techniques? The method was developed by Loeffler and Gaffkey in the laboratory of Robert Koch. The streak plate method depends on

Which organ releases the female gamete under formation, Q. What is the orga...

Q. What is the organ that releases the female gamete under formation? How is this release triggered? What is the organ that collects the released gametes? The organ that libera

What are the parts of the female reproductive system, What are the organs t...

What are the organs that are part of the female reproductive system? The organs that comprise the female reproductive system are the ovaries, the Fallopian tubes (or uterine t

Modern theory in biology, Modern Theory Naturalistic theory: Accordin...

Modern Theory Naturalistic theory: According to this theory Life originated upon our earth spontaneously from nonliving matter. But there are two significant points in this r

Determine the symptoms of dehydration, Determine the Symptoms of Dehydratio...

Determine the Symptoms of Dehydration? The symptoms of dehydration are: Thirst Loss of appetite Decreased urination Impaired physical performance Nau

What is ground meristem in primary growing shoot, What is Ground Meristem i...

What is Ground Meristem in primary growing shoot? The ground meristem produces new cells which ultimately develop into parenchyma cells. The central portion of the primary shoo

Nervvous system, what is the largest nerve centre

what is the largest nerve centre

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