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 is the autophagic intracellular digestion, Q. What is the autophagic i...

Q. What is the autophagic intracellular digestion and why is this type of intracellular digestion intensified in an organism undergoing starvation? Autophagic intracellular dig

What are cells with a delimited nucleus called, What are cells with a delim...

What are cells with a delimited nucleus called ? What are the main elements of the nucleus? Cells with delimited nucleus are known as eukaryotic cells. Organisms composed of on

Tissue, Ask ques different tipe tissue and ideas minimum 100 words accepted...

Ask ques different tipe tissue and ideas minimum 100 words accepted#

#title.classification., merits and demerits system of classification by Lin...

merits and demerits system of classification by Linnaeus

Explain phylum mollusca, Phylum Mollusca (47,000 species) The segmentat...

Phylum Mollusca (47,000 species) The segmentation and coelom may not exist. Diversification through modifications of head, foot; mantle and 'radula for feeding. .Visceralorgans

Why alkaline phosphatases use water as a second substrate, Alkaline Phospha...

Alkaline Phosphatases use water as a second substrate. Why do we still treat their kinetics as if they were for a single-substrate reaction?

Determine the significance of siphuncle., Determine the significance of Sip...

Determine the significance of Siphuncle. A cord of tissue that passes between different chambers of a Nautilid shell. Siphuncle regulates the amount of air or fluid in chambers

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