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

Therapeutics for infective endocarditis, Therapeutic   Accurate identif...

Therapeutic   Accurate identification of the infecting organism is the key to successful treatment.  Antibiotics as per the sensitivity study in high doses IV is given.

Kangroo rat, Kangroo Rat Kangroo rat Dipodornys merriami, a native of ...

Kangroo Rat Kangroo rat Dipodornys merriami, a native of South-West America is a classical example of how small mammals survive in desert. It exhibits all the osmoregulatoryad

Precautions for spore staining in a given bacterial culture, Define some Pr...

Define some Precautions for spore staining in a given bacterial culture? 1. Do not allow malachite green to dry on slide. 2. Decolourization should be done only after coolin

Theory of germplasm, THEOR Y OF GERMPLASM - August Weismann (1834-1...

THEOR Y OF GERMPLASM - August Weismann (1834-1914) criticized the inheritance of acquired characters by putting forward the theory of continuity of germplasm. According

What does boiling do to an enzyme, What does boiling do to an enzyme? Enz...

What does boiling do to an enzyme? Enzymes are denatured (their shape is changed so it doesn't work) when boiled.

Primary egg envelopes, Primary Egg Envelopes These are the egg envelo...

Primary Egg Envelopes These are the egg envelopes which develop in the ovary between oocyte and follicle cells in the space occupied by the interdigitating microvilli. Such e

Maggots and murder, Plot the data as a suitable graph. Join the points with...

Plot the data as a suitable graph. Join the points with the best-fitting smooth curve.Temperature oC(5,10,11,13,17,19) Time taken for eggs to hatch/h 230 90 70 30 25

Explain increases-decreases or shifts in demand terminology, Explain about ...

Explain about the increases or decreases or shifts in demand terminology. Market into equilibrium P 1 Q 1 , here demand D 1 equals supply S 1 • When price reduce fromP 1

Explain night blindness - micronutrient deficiencies, Explain Night Blindne...

Explain Night Blindness - micronutrient deficiencies? Night blindness is the earliest symptom of Vitamin 'A' deficiency. The reduction in the supply of vitamin A aldehyde i.e.

Changes in the conformation - qualitative changes, Changes in the conformat...

Changes in the conformation of molecules - Qualitative Changes You may recall that linear chain of amino acids of a protein folds into a characteristic structure. Acidic resid

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