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 is signifying by suction force of the plant cell, Q. What is signifyin...

Q. What is signifying by suction force of the plant cell? Does the suction force facilitate or make difficult the entry of water into the cell? The suction force (SF) is the os

Plant physiology, why does the removal of extremity of coleoptile prohibit ...

why does the removal of extremity of coleoptile prohibit plant growth?

Micro biology, explane the role of nitrogenase enzyme

explane the role of nitrogenase enzyme

What is the quaternary structure of a protein, What is the quaternary struc...

What is the quaternary structure of a protein? Do all proteins have quaternary structure? The quaternary protein structure is the spatial conformation because of interactions b

Cut-off points to judge on failures - root canal treatment, Determine cut-o...

Determine cut-off points to judge on failures - Root Canal Treatment a)  Persistent of signs and/or symptom. b)  If in one year there is no decrease in lesion size. c)  I

Why isnt the cooking of vitamin c-containing foods, Why isn't the cooking o...

Why isn't the cooking of vitamin C-containing foods appropriate for vitamin C supply? To obtain vitamin C, for example, from an orange dessert, the vitamin-containing food cann

Effective utilization of agro- industrial by- products, Research Priorities...

Research Priorities for Effective Utilization of Agro- Industrial By- Products Biotechnology including recombinant DNA techniques provides for genetic modifications in plants a

Centrioles, Centrioles Centrioles is also called cell centre . C...

Centrioles Centrioles is also called cell centre . Centrosome discovered by Benden . T. Boveri called it centrosome. Centrosome is present in all animal eukaryotic

Syngamy - protozoan, Syngamy - Protozoan The two gametes may be morpho...

Syngamy - Protozoan The two gametes may be morphologically similar (isogametes) or dissimilar (anisogametes). The gametes also vary in form, they may be flagellated or amoeboi

Aetiology, Aetiology:   A List Of The Causes Of A Disease  (Description Of ...

Aetiology:   A List Of The Causes Of A Disease  (Description Of The Characteristic Causes) Factors Which Influence The Onset Of Disease:  Include "Risk Factors", Life-Style, Sex, A

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