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

Amphibia, an assignment of amphibians

an assignment of amphibians

What is biomolecules?, A biomolecule is any molecule that is produced by a ...

A biomolecule is any molecule that is produced by a living organism, including large macromolecules like proteins, polysaccharides, lipids, and nucleic acids, as well as small mole

Assignment, protoplasmic level of oganisation

protoplasmic level of oganisation

Sex-influenced traits, what are the examples of sex-influenced traits in hu...

what are the examples of sex-influenced traits in human?

Explain about the parkinson''s disease, Explain about the Parkinson's disea...

Explain about the Parkinson's disease? Parkinson's disease is a degenerative central nervous system (CNS) condition characterized by progressive loss of cells within substantia

What is the radula, What is the radula? What is the function of this organ?...

What is the radula? What is the function of this organ? Some molluscs have a tongue-like structure with harsh saliences similar to small teeth. This structure is called radula

Explain bacterial vaginosis, Bacterial vaginosis  The role of sexual tr...

Bacterial vaginosis  The role of sexual transmission is unclear in bacterial vaginosis, in which normal H2O2-producing Lactobacillus  species are replaced by overgrowth with  G

Requirements of various nutrients in constipation, Q. Requirements of vario...

Q. Requirements of various nutrients in constipation? The requirements of various nutrients are not altered in constipation. It is essentially a normal balanced diet (normal RD

Explain some feeding considerations or nutrition support, Explain some Feed...

Explain some Feeding Considerations or Nutrition Support? The preferred route for feeding the patient should be oral intake1 via the utilization of gastrointestinal tract. If o

Criticism of darwinism, CRITICISM OF DARWINISM - Arriva l of the f...

CRITICISM OF DARWINISM - Arriva l of the fittest: Darwinism explains the survival of the fittest but was unable to account for the arrival of the fittest. V estigia

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