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
Determine the nutritional needs of humans? In the previous unit, we saw how involvement in sport or vigorous activities can affect the body's nutrient needs. In this unit, we w

What is the role of mineral salts in the creation of electric tension (voltage) at the cellular level? The electric activity of the cell, for instance, in neurons, depends on

As double-stranded DNA is heated a temperature is reached at that the two reaction strands divided.  This procedure is called as denaturation.  The temperature at that half of the

what are the different modes of nutrition

Explain about the Fermentation - methods of food processing? In contrast to other preservation methods, multiplication of microorganisms and their metabolic activities are enco

The use of the gums in various foods may affect the following functional properties: 1. Water-binding capacity 2. Rheological properties 3.  Capacity to form film or gel

What are the main constituents of the earth's atmosphere in our time? The present atmosphere of the earth is constituted mostly of molecular nitrogen (N2) and molecular oxygen


What are melanocytes? Melanocytes are epithelial cells of the skin particular in secretion of melanin. Melanin is a pigment that besides coloring the skin, the iris of the eye

State about polyploid cells During development cells of certain tissues like the salivary gland and liver produce extraordinary amounts of selected proteins. This is often achi