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

State the major function of the cell membrane, State the major function of ...

State the major function of the cell membrane A major function of the cell membrane is to maintain the characteristic integrity of the cell by forming a selective barrier betwe

Necessity of transport in phloem, Necessity of Transport in Phloem Lea...

Necessity of Transport in Phloem Leaves produce photoassimilates and support various tissues including roots. The excess photoassimilates and metabolites stored in specialised

Assistance required, I have been getting assignments to write. But not for ...

I have been getting assignments to write. But not for the subject I have aptitude for.Please help

Explain micafungin, Explain Micafungin It is an echinocandin indicated ...

Explain Micafungin It is an echinocandin indicated for treatment of esophageal candidiasis and prophylaxis of invasive Candida infections in patients undergoing hematopoietic s

State the testing of ketones in urine, State the Testing of Ketones in Urin...

State the Testing of Ketones in Urine Ketones are also known as ketone bodies. Ketone bodies are catabolic products of free fatty acids and three ketone bodies that can be dete

Snail, what are the negative and positive economic importance of snai

what are the negative and positive economic importance of snai

What are nutritional issues related to neurological disorder, What are nutr...

What are nutritional issues related to neurological disorders? Nutritional management of the patients with neurological disease is complex, as mechanisms and abilities needed f

Coral reefs, Coral reefs form in clear warm tropical seas and are particula...

Coral reefs form in clear warm tropical seas and are particularly well developed in the South Pacific. They are formed by accumulation in calcarious skeletons of tiny colonial anim

Explain ledge bypass - non-surgical endodontic retreatment, Explain Ledge b...

Explain Ledge bypass - Non-surgical Endodontic Retreatment   Ledge bypass - StSt not NiT cause it has shape memory Ledges from outside the curvatures. The file t

Explain what is mortality in coronaw artery disease, Explain what is Mortal...

Explain what is Mortality in coronaw artery disease ? Marked prematurity and extensive atherosclerosis leads to markedly higher mortality in young Asian Indians compared to oth

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