User Account

All Pages

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

Determine the long buccal nerve, Long buccal nerve The long buccal nerv...

Long buccal nerve The long buccal nerve is a branch of the mandibular division of the trigeminal nerve which provides sensory innervation to the buccal gingiva and mucosa of th

Define malnutrition - effects on milk and effects on mothers, Define Malnut...

Define Malnutrition - Effects on Milk and Effects on Mothers? Milk is the sole source of nourishment for many infants for upto 6 months or a year or even more. Therefore, the r

Which type of cell came first in evolution, Q. Which type of cell came firs...

Q. Which type of cell came first in evolution - the eukaryotic cell or the prokaryotic cell? This is an interesting problem of biological evolution. The majorly accepted hypoth

Twins, how twins are produced and why

how twins are produced and why

Explain the hormonal control of blood sugar levels, The sugar carried in th...

The sugar carried in the blood is glucose; the normal value of sugar in the human bloodstream is approximately 90mg per 100 bloods. Sugar is absorbed from the small intestines and

What are the three major types of rna, Q. What are the three major types of...

Q. What are the three major types of RNA? What is meant by heterogeneous RNA? Messenger RNA, or mRNA, transfer RNA, or tRNA, and ribosomal RNA, or rRNA, are the three main type

List out the principles of food preservation, List out the Principles of Fo...

List out the Principles of Food Preservation? All food preservation methods are based upon the general principle of preventing or retarding the causes of spoilage caused by mic

What do you mean by herbaria and museums, Q. What do you mean by Herbaria a...

Q. What do you mean by Herbaria and Museums? A herbarium is a collection of pressed and dried plants arranged according to some valid system of classification and available for

Explain the riboflavin content of rbc - riboflavin, Explain the Riboflavin ...

Explain the Riboflavin content of RBC - Riboflavin? The erythrocyte glutathione reductase assay, with an activity coe cient (AC) expressing the ratio of activities in the prese

How to calculate the requirements for the calcium in body, How to Calculate...

How to Calculate the Requirements for the Calcium in Body? Requirements for calcium depend upon the rate at which calcium is incorporated into bone; they are therefore highest

Write Your Message!

Captcha
Order Assignment

Featured Services

Order Now

Popular Subjects

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!

Submit Assignment

Academics

Major Subjects

Majors

Get In Touch

whatsapp: +91-977-207-8620

Phone: +91-977-207-8620

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd