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

Define the reaction to food additives, Define the Reaction to Food Additive...

Define the Reaction to Food Additives? Food additives such as preservatives, flavour enhancers and colouring agents have been linked to adverse food reactions. Additives implic

Explain the stages in taxonomic procedures - alpha taxonomy, Explain the St...

Explain the Stages In Taxonomic Procedures? Divided into three levels or phases: 1) Alpha (α) phase, 2) Beta (β) phase, 3) Gamma (γ) phase. Alpha Taxonomy The

Infective endocarditis, Infective Endocarditis   It  is a term used to ...

Infective Endocarditis   It  is a term used to include subacute bacterial endocarditis as well as infection caused by viruses  and fungi.  The causative organisms responsibl

Proteins, what is he importance of proteins for living beings

what is he importance of proteins for living beings

What mechanism of regulation utilized in maintaining, What mechanism of reg...

What mechanism of regulation is utilized in maintaining homeostatic balance of sugar?

Define isotope dilution method, Define Isotope Dilution Method? The mea...

Define Isotope Dilution Method? The measurement of total body water (TBW) is based on the principle of hydrometry. You must be aware that water is the most abundant substance i

Expression, To "express" a gene is to make it to function properly. A gene ...

To "express" a gene is to make it to function properly. A gene which encodes a protein will, when expressed, be transcribed or translated to produce that particular protein. A gene

Panting - temperature relations in animals, Normal 0 false fa...

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

First stage of invagination, First Stage of Invagination The large en...

First Stage of Invagination The large endodermal cells that are remaining in the vegetal plate move laterally towards the centre of the plate and fill the gap in the vegetal

Phylum annelida, where we find annelid

where we find annelid

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