Cloning protocol, Biology

Assignment Help:

1945_Alkaline Phosphate treatment.png

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.

(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?)


Related Discussions:- Cloning protocol

Show the process by which the neural impulse is transmitted, Q. What is the...

Q. What is the mechanism by which the neural impulse is transmitted along the axon? The neural impulse is transmitted along the neuronal membrane through depolarization of cons

Define reaction of fehling soxhlet method, Define Reaction of Fehling Soxhl...

Define Reaction of Fehling Soxhlet method? The reducing sugars formed are acted upon by the alkali of Fehling's solution to form enediols. These enediols are very unstable and

What is the molecular composition of hemoglobin, What is the molecular comp...

What is the molecular composition of hemoglobin? Does the functionality of hemoglobin as a protein depend upon its tertiary or upon its quaternary structure? Hemoglobin is a mo

Selection of foods for diabetic patients, Q. Selection of foods for diabeti...

Q. Selection of foods for diabetic patients? Some handy guidelines for selection of foods for diabetic patients is presented next. You may be aware that some foods need to be

Discuss the biological concepts of the migration, The bar-tailed godwit, Li...

The bar-tailed godwit, Limosa lapponica baueri, or kuaka, is a wading shorebird seen on marshy estuaries and wetlands during the New Zealand summer. Populations of this species emb

Concomitant caroid endarterectomy and cabg, Concomitant caroid endarterecto...

Concomitant caroid endarterectomy and CABG :   Symptoimatic or asymptomatic carotid artery disease may be present in patients undergoing CABG. It is very important to recognize thi

Explain about the various cooking methods, Explain about the various Cookin...

Explain about the various Cooking methods? All of us eat food either raw or in cooked form. Have you ever thought why we need to cook food?  Cooking is a primary process to mak

Anatomy of the brain, In order to be able to study the neural basis of perc...

In order to be able to study the neural basis of perceptual and cognitive functions, it is necessary to have some knowledge of brain anatomy (i.e., what the names of different brai

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