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

Dermatitis, D i se a se s of Skin Der m a t i t i s De...

D i se a se s of Skin Der m a t i t i s Dermatitis is the inflammation of dermis and epidermis resulting in pain on palpation and erythema. E t iolo

Use of genital herpes in pregnancy, Use of genital herpes in pregnancy ...

Use of genital herpes in pregnancy Although acyclovir is not approved for treatment of pregnant women, its use during pregnancy has not been associated with an increased risk

Define the interaction of vitamin c with iron, Define the Interaction of Vi...

Define the Interaction of Vitamin C with iron? Ascorbic acid powerfully enhances absorption of non- haem iron and reverses the inhibiting effect of tea and calcium phosphate. I

Neurosecretory systems, Neurosecretory Systems Almost all groups of m...

Neurosecretory Systems Almost all groups of multicellular animals have been investigated to find out if neurosecretory cells are present in them. Such investigations have sho

Signs and symptoms of emphysema, Pathophysiology Distension and damage...

Pathophysiology Distension and damage of the respiratory bronchioles selectively occur. Opening develops in the walls of the bronchioles. and they become enlarged and conf

Why heartwood differs from sapwood, Heartwood differs from sapwood in: 1...

Heartwood differs from sapwood in: 1. Presence of rays and fibres 2. Absence of vessels and parenchyma 3. Having dead and non-conducting elements 4. Being susceptible t

What is the vacuum puff drying, What is the Vacuum Puff Drying? The vac...

What is the Vacuum Puff Drying? The vacuum puff process has been developed for drying liquids under vacuum. The development of processes for drying liquids under vacuum came fr

What is an accommodation, What is an accommodation? Accommodation is th...

What is an accommodation? Accommodation is the ability of the eye to focus on an object situated at variable distances from the eye. Like a camera, even the eye requires to cha

Structure of the sperm, Structure of the Sperm The spermatozoa in dif...

Structure of the Sperm The spermatozoa in different animal groups display a wide variety of shapes and sizes but all share a. basic morphological plan. The sperm in most anim

What is the importance of pre procedural rinse, Q. What is the importance o...

Q. What is the importance of pre procedural rinse? Patients' use of an anti-microbial mouthwash of 0.12% chlorhexidine gluconate solution for 30 seconds prior to intra-oral pro

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