Cloning protocol, Biology

Assignment Help:

1945_Alkaline Phosphate treatment.png

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.

(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?)


Related Discussions:- Cloning protocol

List the types of gellan gum, List the types of Gellan gum.   The three...

List the types of Gellan gum.   The three types of gellan gums are: High acetyl gellan (partially deacetylated) Low acetyl gellan (highly deacetylated) High cl

Central and peripheral nervous system - geriatric nutrition, Explain Centra...

Explain Central and Peripheral Nervous System - Geriatric Nutrition? Age related changes occur in various senses related to the cranial nerves-vision, hearing, taste and smell

Define self monitoring of blood glucose, Q. Define Self Monitoring of Blood...

Q. Define Self Monitoring of Blood Glucose? Self Monitoring of Blood Glucose (SMBG): A patient can monitor the glucose level at home by self using the glucometer for checking t

What is vernalisation, What is vernalisation? How is the process of vern...

What is vernalisation? How is the process of vernalisation benefits to plants?

Develop a hypothesis relating to the amount, Develop a hypothesis relating ...

Develop a hypothesis relating to the amount of dissolved oxygen measured in the water sample and the number of fish observed in the body of water.

Theory of embryology - theory of epigenesis, THEOR Y OF EPIGENESIS - ...

THEOR Y OF EPIGENESIS - It was propounded by C. F. Wolff and supported by Von Baer. According to it, the egg contains the substances which are required in the formation

Staphylococcal food poisoning, Staphylococcal food poisoning results from c...

Staphylococcal food poisoning results from consumption of food containing enterotoxin produced by enterotoxigenic strains of Staphylococcus aureus. It is caused by ingestion of imp

What is glycogen metabolism, Glycogen is a multibranched polysaccharide whi...

Glycogen is a multibranched polysaccharide which serves as a part of energy storage in fungi and animals. In humans the glycogen is recognized and stored primarily in the cells of

The statement is true or false, Anton van Leeuwenhoek was the first to prop...

Anton van Leeuwenhoek was the first to propose placing microorganisms into the Kingdom Monera. True or False

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