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

Define role of lipids in controlling gene expression, Define role of Lipids...

Define role of Lipids in Controlling Gene Expression? In the lipogenic pathway, the genes that encode the enzymes of the fatty acid synthase complex are co-ordinately expressed

Use of computer in biotechnology, what are the uses of computer in biotechn...

what are the uses of computer in biotechnology ??? briefly explain any twenty

Golgi apparatus, GOLGI APPARATUS Camillo Golgi in 1898 discovered a retic...

GOLGI APPARATUS Camillo Golgi in 1898 discovered a reticular structure in the cytoplasm of nerve cells with the help of metal impregnation technique using silver nitrate for whic

Kinase, A kinase is in general an enzyme which catalyzes the transfer of th...

A kinase is in general an enzyme which catalyzes the transfer of the phosphate group from ATP to something else. In the molecular biology, it has acquired the more specific verbal

Which molecule should be most radioactive, Yeast is cultured in the presenc...

Yeast is cultured in the presence of radioactive phosphate and the following biological molecules are purified. Which molecule should be most radioactive? A. An oligosaccharide B.

Classify bones structurally and embryologically, Classify bones structurall...

Classify bones structurally and embryologically(developmentally). Structural classification of bone: Macroscopically can be classified into two types, compact and trabecul

Use of syphilis in pregnancy, Syphilis in Pregnancy Syphilis in pregna...

Syphilis in Pregnancy Syphilis in pregnant women should be treated with penicillin in doses appropriate to the stage of the disease. When pregnant women with syphilis are alle

Cloning of arg3 gene, 1)  You want to clone the arg3 gene, which encodes an...

1)  You want to clone the arg3 gene, which encodes an enzyme in the arginine biosynthetic pathway in Neurospora crassa.  You have available the E. coli plasmid vector shown in part

Define nervous system and the it's related disorders, Define Nervous System...

Define Nervous System and the it's related disorders? In this unit, we learnt about nervous system and the related disorders, which are termed as 'neurological disorders'. Neur

Explain about hypoglycemia, Q. Explain about Hypoglycemia? It is a Gree...

Q. Explain about Hypoglycemia? It is a Greek term: hypo -meaning less; glyc- means sweet; and emia- means "of the blood". It is a condition in which less than normal amount of

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