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

Biota of littoral zone, Biota of Littoral Zone This zone is the shore ...

Biota of Littoral Zone This zone is the shore region of the marine ecosystems and is subject to violence of waves and tides, fluctuation of water level and variability of temp

Metabolism of lipids, The three main forms of fat found in food are glyceri...

The three main forms of fat found in food are glycerides (principally triacylglycerol [triglyceride], the form in which fat is stored for fuel), the phospholipids, and the sterols

What is saliva, What is Saliva Saliva   is  the colourless  viscous  f...

What is Saliva Saliva   is  the colourless  viscous  fluid.  It  helps  in  swallowing the  food  by lubricating the food and the neutral pH of saliva prevents the decalcifica

How to repair the defects in post myocardial , How to repair the defects in...

How to repair the defects in post myocardial ? Repair of Defect :  The approach to the defect is through ventriculotomy over the infarcted area. The defect is repaired

Explain corrective surgery - endodontic surgery, Explain Corrective Surgery...

Explain Corrective Surgery - Endodontic Surgery perforation repair periodontal management (root resection, tooth resection) intentional reimplantation

State tantalum and niobium in alloplastic implant material, Tantalum and Ni...

Tantalum and Niobium in alloplastic implant materials  Tantalum has been used successfully in needle type, screw- type and double-bladed implants. However, both are difficult

What is the relevance of knowing about gi of foods, What is the relevance o...

What is the relevance of knowing about GI of foods? Let us see how this index is helpful in prescribing therapeutic diets. You would realize that the dietary GI provides an ind

Explain limited mucosal contact diarrhoea, Q. Explain Limited mucosal conta...

Q. Explain Limited mucosal contact diarrhoea? Limited mucosal contact diarrhoea: It results from situations of inadequate mixing of chyme (semi-liquid mass of food passing thro

Investigation of tricuspid stenosis by echocardiography, Q. Investigation o...

Q. Investigation of Tricuspid stenosis by Echocardiography? Findings are subtle. One should carefully search for organic tricuspid valve involvement in all rheumatic heart dise

Diversity in living organisms, scientific names of animals

scientific names of animals

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