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

Muscular force, 2.?In order to increase muscular force production, muscular...

2.?In order to increase muscular force production, muscular contractions may be modulated in two ways. Explain.

Why water has key participation in organic reactions, Water has key partici...

Water has key participation in organic reactions. What are examples of two types of organic reactions in which water is respectively incorporated or liberated in the products of th

What do you mean by the spinal cord, Q. What is the spinal cord? Of which e...

Q. What is the spinal cord? Of which elements is the spinal cord constituted? The spinal cord is the dorsal neural cord of vertebrates it is the part of the central nervous sys

Define classification of proteins on the basis of attributes, Define Classi...

Define Classification of proteins on the basis of attributes? Besides classifying proteins on the basis of soluble and insoluble, proteins have been further classified based on

A dihybrid cross yields 320 f2 offspring, A dihybrid cross yields 320 F2 of...

A dihybrid cross yields 320 F2 offspring. How many are expected to resemble the homozygous recessive parental?

Does the environment exert an influence on the phenotype, Does the environm...

Does the environment exert an influence on the phenotype? The phenotype may be altered (compared to the original situation conditioned by its genotype) by nongenetic means, ins

Zoonoses disease-staphylococcosis, Staphylococcosis The disease is caused ...

Staphylococcosis The disease is caused by the ingestion of preformed toxin released by Staphylococcus aureus. Epidemiology: Staphylococci grow in meat, dairy and bakery prod

Draw a diagram of transverse section of mammalian bone, Draw a diagram of t...

Draw a diagram of transverse section of mammalian bone and label the following parts in it: (i) Haversian canal (ii) Lacuna containing osteocytes (iii) Canaliculi

Diaphragmatic hernia, Diaphragmatic Hernia: In this condition there  i...

Diaphragmatic Hernia: In this condition there  is a slight herniation ofabdominal organs  (stomach intestine and liver) or extreme protrusion of abdominal contents into the th

Define the nutritional and functional role of iodine, Minerals :- Iodine  ...

Minerals :- Iodine  Food Source      Iodised salt, sea food, plants and animals grown in areas where soil iodine is not depleted. Nutritional Functional role Esse

Write Your Message!

Captcha
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!

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd