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

Define prevalence of weight management, Define Prevalence of Weight Managem...

Define Prevalence of Weight Management? WHO (1998) estimates that in developing countries about 245 million adults are moderately underweight and 93 million severely underweigh

Right coronary artery, Right coronary artery arises from the anterior aorti...

Right coronary artery arises from the anterior aortic sinus. It runs down along the anterior part of the coronary sulcus, reaches the lower border of heart turns round the lower bo

Public-private dichotomy in providing health services, Normal 0 ...

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

How are bacteria are classified, Q. How are bacteria are classified accordi...

Q. How are bacteria are classified according to their need for oxygen? According to their need of oxygen bacteria are classified into anaerobic those that survive without oxyge

Does the molecule will remain unchanged, A double-stranded piece of DNA, 10...

A double-stranded piece of DNA, 100 base pairs in size, is exposed to a high pH environment. This causes all non-valent interactions to be disrupted. What will result from this tre

Respiration – protozoan, Respiration – Protozoan Gas exchange occurs b...

Respiration – Protozoan Gas exchange occurs by the diffusion of oxygen across the cell membrane. Some protozoan utilize this oxygen but are also capable of anaerobic respirati

What do you mean by canning, Q. What do you mean by Canning? Canning T...

Q. What do you mean by Canning? Canning The term canning is generally applied to foods, more specifically, to the foods preserved by heat processing. It aimgto destroy microo

Illustrate the important rules of nomenclature, Q. Illustrate the Important...

Q. Illustrate the Important rules of nomenclature? Nomenclature is allied to taxonomy as it deals with the determination of the correct name to be applied to a known taxon. The

Define important food sources of protein, Define Important Food Sources of ...

Define Important Food Sources of Protein? The important sources of proteins in the diets of low-income groups are cereals and legumes. Meat, fish, eggs and milk are important s

Define dissection of the aorta, Q. Define Dissection of the Aorta ? The...

Q. Define Dissection of the Aorta ? The CXR may be abnormal in upto 80 per cent of patients. The abnormalities include: i) Widened mediastinum: This is defined as a mediast

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