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

What are the segments that form the body of the tapeworm, What are the segm...

What are the segments that form the body of the tapeworm called? What is their function? The body of the tapeworm is made of segments known as proglottids. The proglottids are

Will any cell verify the cell theory, Of the cell types observed (Parameci...

Of the cell types observed (Paramecium, Euglena, Yeast, Elodea, cheek cells), would any cell "verify" the cell theory? Explain your answer.

Green glands of crustaceans, Normal 0 false false false ...

Normal 0 false false false EN-IN X-NONE X-NONE MicrosoftInternetExplorer4

Objectives of taxonomy, Q. Objectives of taxonomy? Plants and animals b...

Q. Objectives of taxonomy? Plants and animals both have a great significance and unparalleled impact on human life. Most of our needs are fulfilled with plants such as foodstuf

Cytology, who is the first person who saw the cell

who is the first person who saw the cell

Clinical symptoms of hiv, Clinical symptoms of HIV Generally, a person ...

Clinical symptoms of HIV Generally, a person infected with HIV does not show any apparent symptoms for a number (3 to 12) of years. Ten to fifteen percent infected people may,

How to compare and contrast acids, Compare and contrast acids and bases in ...

Compare and contrast acids and bases in terms of their H+ ion and OH- ion concentrations.

Products of cleavage - morula and blastula, Products of Cleavage (Morula an...

Products of Cleavage (Morula and blastula) In several cases, the blastomeres in early cleaving stages tend to presume spherical shape like that of the egg earlier than cleava

What is the importance of vitamin B6, What is the Importance of vitamin B 6...

What is the Importance of vitamin B 6 In the human and animal organisms, vitamin B 6 acts in the form of pyridoxal. In the form of 5'-orthophosphate (Pyridoxal-5'-phosphate),

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