User Account

All Pages

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 the name of surgical needle, Illustrate the name of surgical ne...

Illustrate the name of surgical needle The surgical needle is comprised of 3 parts: the needle point, the needle body, and the swaged (press-fit) end. Needle may be broadl

..kingdom animalia, Rat physical characteristics

Rat physical characteristics

Describe congenital pulmonary stenosis, Describe Congenital Pulmonary Steno...

Describe Congenital Pulmonary Stenosis? The murmur present since birth. PS may be valvular, sub-valvular, or supravalvular. An obstruction may occur within the RV cavity by an

Classification of proteins based on chemical properties, Classification of ...

Classification of proteins based on chemical properties? Depending on their chemical properties and optical activity, the amino acids in proteins are classified under the follo

Nature defences against viral infections, Nature defences against viral inf...

Nature defences against viral infections 1.      Interferons - In 1957 Issue and Lindenmann discovered that vertebrate cells infected with viruses, produce a diffusible antiv

Why do some physicians advise patients, Why do some physicians advise patie...

Why do some physicians advise patients with a bladder infection to drink a lot of cranberry juice? Answer Cranberry juice contains chemicals that kill viruses and bacteria. The inc

How do sponges try to protect themselves against harm, How do sponges try t...

How do sponges try to protect themselves against harm from the environment? Is that method efficient or rudimentary? Sponges can close their pores to avoid the entrance of wate

Explain about phylum annelida, How is the respiratory system of beings of ...

How is the respiratory system of beings of the phylum Annelida characterized? Respiration in annelids can be cutaneous or branchial. Cutaneous respiration happens due to the r

Drawbacks of free food items for diabetics patients, Drawbacks of Free Food...

Drawbacks of Free Food Items for diabetics patients In this context, it is important to note that a patient can have unlimited servings of 'free foods'. These are foods which d

Roles of fatty acid, Fatty acids have four main biological roles: 1.  Th...

Fatty acids have four main biological roles: 1.  They are used to create sphingolipids and glycerophospholipids which are necessary elements of biological membranes; 2.  Nume

Write Your Message!

Captcha
Order Assignment

Featured Services

Order Now

Popular Subjects

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!

Submit Assignment

Academics

Major Subjects

Majors

Get In Touch

whatsapp: +91-977-207-8620

Phone: +91-977-207-8620

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd