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

What are heterochromatin and euchromatin, Chromatin is uncondensed nuclear ...

Chromatin is uncondensed nuclear DNA, the typical DNA morphology in interphase (the phase of the cell cycle in which the cells is not separating itself). In this phase of the cell

Define alanine cycle, Alanine cycle Alanine Cycle  :  In the alanine ...

Alanine cycle Alanine Cycle  :  In the alanine cycle the pyruvate formed from glycolysis  in  the muscle is converted  to alanine by transamination  reaction. Alanine is rele

Basic concepts of earthquake resistant structures, The aim of earthquake-re...

The aim of earthquake-resistant structure designing The main objectives of earthquake resistant structures are as follows: (i)     To avoid loss of lives due to the destruct

Explain human fetal circulation and the adult circulation, Concerning the m...

Concerning the mixture of arterial with venous blood what is the difference between the human fetal circulation and the adult circulation? In the human fetal circulation there

Flow cytometry, The following histograms were produced using flow cytometry...

The following histograms were produced using flow cytometry after labelling B-cell lymphocytes with propidium iodide. Histogram A is from a healthy individual while Histogram B is

Illustrate the types of Bone Remodeling, Illustrate the types of Bone Remod...

Illustrate the types of Bone Remodeling Bone remodeling occurs on existing bone surfaces. However, unlike modeling, remodeling cannot cause large changes in bone structure at a

Define characteristics of phylum nematode, Review of Characteristics of Phy...

Review of Characteristics of Phylum Nematode and Its Position in Animal Classification? Nematodes may be free-living or parasites of plants or animals. however, all nematode

Explain the type I diabetes mellitus, What the difference is between type I...

What the difference is between type I diabetes mellitus and type II diabetes mellitus? Type I diabetes, also called as juvenile diabetes, or insulin-dependent diabetes (this na

ANIMAL RESPIRATION, WHAT IS THERE BETWEEN THE TRACHEA OF COCKROACH AND RABB...

WHAT IS THERE BETWEEN THE TRACHEA OF COCKROACH AND RABBIT

Explain uristix benedicts test, Q. Explain Uristix Benedicts test? No s...

Q. Explain Uristix Benedicts test? No sugar/glucose is excreted in the urine in normal individuals. In diabetics, glucose is excreted by the kidneys when the blood sugar levels

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