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

Method of purchasing a service, Normal 0 false false false ...

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

Discuss how would you perform antiglobulin test, Question 1 Discuss how wo...

Question 1 Discuss how would you perform antiglobulin test? Add a note on it clinical importance. What are the precautions to be taken while performing the test? Question 2 E

Xylem transfer cells, Xylem transfer cells The lateral transport of io...

Xylem transfer cells The lateral transport of ions from root xylem to leaves probably takes place via xylem transfer cells which have two special features: The ce

What is the significance of the epiglottis in human body, What is the signi...

What is the significance of the epiglottis in human body? What happens to the glycogen concentration in the liver cells when the level of adrenaline enhances in the blood strea

Explain mendels law of dominance, Which one of the following cannot be expl...

Which one of the following cannot be explained on the basis of Mendel's Law of Dominance? 1. The discrete unit controlling a particular character is called a factor 2. Out o

Nutrition in animals, composition,sources,importance,types,requirements for...

composition,sources,importance,types,requirements for each food substances

Cancer lung, Cancer Lung Incidence of cancer lung is more in persons ...

Cancer Lung Incidence of cancer lung is more in persons who smoke. Environmental and industrial pollution is also a risk factor in the increase in incidence. Clinic

Describe the roles and mechanisms of these rna molecules, 1 ." Most of the ...

1 ." Most of the genome does not code for protein, therefore 99% of the human genome is junk that serves no purpose". With specific reference to examples long and short non-coding

Etiologic factor of atherosclerosis, Q. Etiologic factor of atherosclerosis...

Q. Etiologic factor of atherosclerosis? Various factors are responsible for atherosclerosis. These include: 1. Hyperlipidemin: Excess circulating fats in blood especially th

Discuss in brief about headaches, Discuss in brief about the Headaches ...

Discuss in brief about the Headaches Headache is so common among the general population that rare indeed is the person who has never suffered one. Headache may constitute a neu

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