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

Determine the enzymes utilization in food industry, Enzymes utilization in ...

Enzymes utilization in food industry  Enzymes may be used in industry as components of living cells or after isolation in free or immobilized forms. All of them may be referred

Kingdom animalia, in what order did the shellfish belong? in what genus did...

in what order did the shellfish belong? in what genus did the shellfish belong? in what species did the shellfish belong?

Explain the salient feature of genetic code, The one aspect which is not a ...

The one aspect which is not a salient feature of genetic code, is its being: 1. Degenerate 2. Ambiguous 3. Universal 4. Specific Ambiguous

Explain the term- pupil, Explain the term- PUPIL Pupil is  the  apertu...

Explain the term- PUPIL Pupil is  the  aperture in  centre of  the  iris diaphragm that regulates entry  of  light into the eye. Colour of  pupil  is  said  to be  black. Actu

Electrocardiography, E l ec t r o c ar d i og...

E l ec t r o c ar d i og r ap h y : This equipment is used to identify cardiac conduction abnormalities that limit heart perf

Can you explain bauhin binomial system, Q. Can you explain Bauhin? We g...

Q. Can you explain Bauhin? We got the first reference of binary use of species name in Pinax (1623), a publication of a Swiss physician and botanist Casper Bauhin (1560-1624).

Biocontrol agents , Use of micro-organisms to control insect pests, pathog...

Use of micro-organisms to control insect pests, pathogens or weeds constitutes biological control, and the biological agents employed are called biocontrol agents. Micro-organis

Define the genetic growth pattern of infant, Define the genetic growth patt...

Define the genetic growth pattern of infant? It takes 1-3 years for an infant to establish his own genetic growth pattern. Once this figure is established (such as height-for-a

Explain neurosyphilis, Neurosyphilis  Symptomatic neurosyphilis, includ...

Neurosyphilis  Symptomatic neurosyphilis, including ophthalmic or otic infection, requires treatment with high doses of aqueous penicillin G IV or procaine penicillin G IM with

Explain the five kingdom systems, The five kingdom systems To cope up w...

The five kingdom systems To cope up with above discussed problem a number of alternative classificatory schemes have been suggested with more than two kingdoms. The one which h

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