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
The part of human brain which is a significant relay station for the sensory impulses and also is the origin of many of the involuntary acts of eye such as narrowing of the pupil i

Q What are the most tremendous known representatives of the platyhelminth phylum? The most popular representatives of the platyhelminthes are worms that cause human diseases, l

Problems of Osmoregulation The water, some inorganic salts, nutrient molecules are some of the important components of the body fluids. It is essential for the animals to main

Evaluation of the programme or agency: It can be  based on following:  i)   Audit or review of programme or agency: This can be continuously done by the staff through in

Jillian and Michael are in their freshman year at college. The two have been friends since grade school and the two of them enjoy getting together for dinner. Michael typically

Define the Starch gel electrophoresis? Starch gel has the advantage of fine texture and absence of protein adsorption with a molecular sieving effect retarding proteins of larg

Quantitative changes The quantitative changes in the availability of the active biomolecules occur in order to compensate for the loss of efficiency of the system caused by th

Determine about the disease - brain tumor Tumor, or neoplasm, is a mass of new tissue that persists and grows independently of its surrounding structures and has no physiologic

What are the main substances transferred from the mother to the fetus through the placenta? And from the fetus to the mother? From the mother to the fetus the major transferre

Coelomoducts of Molluscs In Molluscs, like in Crustacea nephridia are absent. But certain of the larval pulmonates do possess protonephridia suggesting that they have been se