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

State the principle of adaptation, State the Principle of Adaptation Th...

State the Principle of Adaptation The first principle is that the central goal of assessment is to promote the adaptation of the child, rather than simply to document the prese

Give detail explanation about swimming, Give detail explanation about Swimm...

Give detail explanation about Swimming Swimming is an excellent form of exercise. Swimming can improve the posture and circulation. It is a healthy activity. There are certain

Nucleic acid, separation of nucleic acid

separation of nucleic acid

Concept of recombination frequency used in genetic mapping, How can the con...

How can the concept of recombination frequency be used in genetic mapping? The Genetic mapping is the determination of the location of the genes in a chromosome. Besides determ

Which areas of the globe is gymnosperm abundance noteworthy, Q. In which ar...

Q. In which areas of the globe is gymnosperm abundance noteworthy? These plants are the typical vegetation of cold regions like the boreal forest or taiga of the northern hemis

Assignment of botany, habitat, habit, cell wall chemistry, reason why cell ...

habitat, habit, cell wall chemistry, reason why cell wall like this of Archaebacteria , eubacteria,fungi,algae,bryophytes ,pteridophyes, gymnosperms , angiosperms..

Evolution, when does it started?

when does it started?

Explain in detail about the concomitant squint, Explain in detail about the...

Explain in detail about the Concomitant squint Concomitant squint is also called comitant squint. It means "I follow you", i.e., in all directions of gazes, the squinted eye fo

Explain about the browning reactions, Explain about the Browning Reactions?...

Explain about the Browning Reactions? The Browning Reactions are complex reactions which occur when foods are processed. In some the brown flavour is highly desirable and is in

Describe systolic sounds in heart dieases, Describe Systolic Sounds in hear...

Describe Systolic Sounds in heart dieases? Early Systolic Sounds: Aortic or pulmonary ejection sounds are the most common early systolic sounds. These are high frequency soun

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