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

Disposal-lab animals, Disposal : Opaque bags are the recommended for dispo...

Disposal : Opaque bags are the recommended for disposing off carcasses. You should also put freshly dissected animals and tissues into opaque plastic bags, seal them and dispose t

Define the term- life on earth, The article entitled "Life on Earth" is try...

The article entitled "Life on Earth" is trying to make the point that RNA likely included before DNA. Forthis article which of the following is false? A. Chains of RNA nucleoti

Describe the number of implants, Number of Implants The use of more imp...

Number of Implants The use of more implants decreases the number of pontics and the associated mechanics and strains on the prosthesis, and dissipates stresses more effectively

Is there a respiratory pigment in the annelid blood, Is there a respiratory...

Is there a respiratory pigment in the annelid blood? The blood in beings of the phylum Annelida have the respiratory pigment hemoglobin (the similar found in chordates) and oth

Endomitosis, why and where endomitosis happen.........?

why and where endomitosis happen.........?

Polymorphism in lysosomes, Polymorphism in Lysosomes Polymorphism, i.e. e...

Polymorphism in Lysosomes Polymorphism, i.e. existence of a structure in more than one form, is an important feature of lysosomes. Several different forms of lysosomes have been

Scintigraphy or nuclear medicine, S c inti g r ap h y ...

S c inti g r ap h y or nuclear medicine: Scintigraphy was introduced in veterinary medicine in 1970s. It is highly sensitive and can detect bone dama

How inadequate breast milk cause protein energy malnutrition, How Inadequat...

How Inadequate Breast Milk cause protein energy malnutrition? Though prolonged breastfeeding of children is the rule in US, the amount of breast milk secreted in poor Indian mo

Complete feed system in ruminants, Complete feed system in ruminants C...

Complete feed system in ruminants Complete feed system for feeding dairy animals is a departure from conventional individual rationing method. It involves the self-feeding of

Nucleolus, NUCLEOLUS Discovered by Fontana and name given by Bowm...

NUCLEOLUS Discovered by Fontana and name given by Bowman. It is naked structure that is attached to chromatin at specific point called nucleolar organiser region or

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