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

Illustrate chloroplast structure, Q. In which chloroplast structure are chl...

Q. In which chloroplast structure are chlorophyll molecules found? Chlorophyll molecules are placed in an organized manner in order to improve the exposure to light on the thyl

The best absorbed in an acidic environment, Why is a weekly acidic drug bes...

Why is a weekly acidic drug best absorbed in an acidic environment?

Sexual reproduction, SEXUAL REPRODUCTION You must be aware that reproduct...

SEXUAL REPRODUCTION You must be aware that reproduction is one of the distinguishing features of living beings. Reproduction is basically of two types: asexual and sexual. In ase

importance of water in the body, Importance of water in the body Wa...

Importance of water in the body Water is the most essential constituent of life. Life cannot exist without water. About 90% of water is present in protoplasm  Water

Loading and unloading of sieve tubes, Loading and Unloading of Sieve Tubes ...

Loading and Unloading of Sieve Tubes In order to understand the loading of food from manufacturing leaf cells to sieve tubes we must examine the anatomy of a minor vein shown

Pulmonary valvotomy on cardio pulmonary bypass, Pulmonary Valvotomy on ...

Pulmonary Valvotomy on Cardio Pulmonary Bypass :  Patient is connected to cardio pulmonary bypass using ascending aortic cannula and single 01- double (preferred) ven

What factors influence whether a person will develop cancer, What factors i...

What factors influence whether a person will develop cancer? Factors contain the person's age, number of exposures to carcinogens, amount of carcinogen in every exposure, and p

Cell division, What is telokinesis and how is it different in animal then p...

What is telokinesis and how is it different in animal then plant cells?

Percentage ratio of ash in honey, Q. Percentage ratio of ash in honey? ...

Q. Percentage ratio of ash in honey? Standards allow ash content in honey up to 0.5 per cent, but the ash of genuine honey seldom exceeds 0.35 per cent. It is determined by cha

Asymmetrical and spherical - metazoa, Asymmetrical and Spherical - Metazoa ...

Asymmetrical and Spherical - Metazoa Some creatures are asymmetrical: no matter which way we try to divide them through the middle, no two halves would appear alike. In simple

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