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

Explain the modern trends in animal taxonomy, Explain the Modern Trends in ...

Explain the Modern Trends in Animal Taxonomy? Earlier you learnt how taxonomy is interrelated to other biological fields. You also learnt how information is used from other f

Phylum:Echinodermata , What classifies an animal into the Echinodermata phy...

What classifies an animal into the Echinodermata phylum?

Enzyme, name of enzyme that mostly used in industrial biotechnology?

name of enzyme that mostly used in industrial biotechnology?

Scientific activity, describe the factors which decide the broad area of sc...

describe the factors which decide the broad area of scientific activity?

What is oxytocin receptors, Which of the following serves as an actuating s...

Which of the following serves as an actuating signal, or as part of an actuating signal, in a positive feedback system? A. Blood plasma levels of PTH (Parathryroid Hormone).

Neutral fat & oils, Neutral fat & oils These are triglyceryde compou...

Neutral fat & oils These are triglyceryde compounds. 1 molecule of neutral fat consists of 1 molecule of glycerole and 3 molecule of fatty acids. 3 ester bo

Which are the structures that are part of the nervous system, Q. Which are ...

Q. Which are the structures that are part of the nervous system? The structures that form the nervous system can be divided into the peripheral nervous system (PNS) and the cen

Skeleton, biological significant of a skeleton

biological significant of a skeleton

Emboly movement - involution, Emboly Movement - Involution It den...

Emboly Movement - Involution It denotes turning in or rolling over. Involution of mesodermal blastomeres has been observed in Amphioxus, reptiles, monotremes, amphibians,

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