Cloning protocol, Biology

1945_Alkaline Phosphate treatment.png

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.

(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?)

Posted Date: 3/11/2013 1:29:22 AM | Location : United States







Related Discussions:- Cloning protocol, Assignment Help, Ask Question on Cloning protocol, Get Answer, Expert's Help, Cloning protocol Discussions

Write discussion on Cloning protocol
Your posts are moderated
Related Questions
Q. What is xaxim? Most pteridophytes have subterraneous stems similar to the substrate called as rhizomes. Xaxim is a kind of pteridophyte with an aerial stem in generally perp

Explain about the Energy Requirements in elderly? Based on the Benedict - Harris equation for men and women, the daily energy requirements can be calculated. For Men: 66 t

Define the symptoms in Bone marrow of pernicious anaemia? The nucleated red cells of the marrow are greatly increased.  The successive nucleated cell stages in erythropoiesis a

Suggest a mechanism that would allow a plant to grow better if it had intermorph neighbors than if it had intramorph neighbors. Think about the degree of genetic similarity between

Subphylum Sarcodiha Pseudopodia typically present; flagella present in developmental stages of some species; free living or parasitic. Superclass - Rhizopoda Loc

Q. How many ATP molecules are produced for every glucose molecule used in fermentation? And How many ATP molecules are produced for each glucose molecule used in aerobic respiratio

Types of overpopulation - Consumption overpopulation Consumption overpopulation is a phenomenon seen in technologically advanced and affluent countries such as U.S.A., Japan,

How does being able to reproduce asexually benefit the plant?

Energy from water or hydropower            Hydroelectricity is the energy produce from the kinetic energy of water falling from height. Approximately 25% of world electricity

A solid compound was found to contain 42.11% C, 51.46%O and 6.43% H by weight. The molecular weight of the compound was 341. What is the molecular formula of the compound?