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

Disorders of motor neurons and the spinal cord, Disorders of motor neurons ...

Disorders of motor neurons and the spinal cord A number of movement disorders are produced by damage either to the spinal cord or to cortical projections to the spinal cord. Th

Explain major role in contributing towards motivation, Major role in contri...

Major role in contributing towards motivation. Psychological factors: Depression, anxiety or phobia induced by illness,  life- style changes or medication effects, may hinder

Define miscibility in binary liquid systems, Define Miscibility in binary l...

Define Miscibility in binary liquid systems? When two different pure liquids are unable to mix in all proportions, they are said to be partially miscible. When these liquids ar

Chromosomes and chromatin strands are present in stage, Drosophila has 4 bi...

Drosophila has 4 bivalents (homologous chromosomes) formed at Meiosis I. How many chromosomes and chromatin strands are present in each of the following stages- Anaphase of mito

Digestion, Adsk question #Minimum 100 words accepted#saliva enzyme

Adsk question #Minimum 100 words accepted#saliva enzyme

Circulation of nutrients in the coastal zone, Circulation of nutrients in t...

Circulation of nutrients in the coastal zone Circulation of nutrients from the sea bottom to the upper surface, occurs in the coastal regions by two processes Upwelling

Explain the functionality of central nervous system, Explain the functional...

Explain the functionality of Central Nervous System ? The central nervous system is the center of the nervous system, receiving impulses from, and sending impulses to, the per

Explain animal sources of natural colourants, Explain Animal sources of Nov...

Explain Animal sources of Novel Sources of Natural Colourants   The most common animal pigments of use as food colourants are those based on the haem structure. In nature, ha

Taxonomy, classification of grantia

classification of grantia

Respiration, Can you define respiration in 5 oganisms

Can you define respiration in 5 oganisms

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