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

Illustrate the applications of gellan gum, Illustrate the Applications of G...

Illustrate the Applications of Gellan gum Gellan is a highly versatile food ingredient and has numerous applications in foods. The combination of gelatin with gellan, by compar

Explain pathology of aortic stenosis, Q. Explain Pathology of aortic stenos...

Q. Explain Pathology of aortic stenosis? Congenital abnormalities of aortic valve may lead to unicuspid aortic valve. This may be dome shaped with central stenotic orifice. Uni

Aspirin, ASPIRIN - It is acetylsalicyclic acid (C 6 H 4 (OCOCH) 3 ...

ASPIRIN - It is acetylsalicyclic acid (C 6 H 4 (OCOCH) 3 CH 3 ). It changes in salicyclic acid in body. It is analgesic, antipyratic (fever reducing), anti inflamma

Bacterial diseases-actinomyces, Actinomyces Members of the genus Actin...

Actinomyces Members of the genus Actinomyces are facultative anerobic, gram-positive, non acid-fast, non-spore forming, nonhaemolytic rods. All Actinomyces require rich media

Estimation of depth of burn injury, Estimation of Depth of Burn Injury: ...

Estimation of Depth of Burn Injury: A thermal injury is described as partial thickness or full thickness, depending on the depth  and  severity of tissue damage a) First D

State the definition of soil, State the definition of soil The definiti...

State the definition of soil The definition of soil has changed a great deal from a thin "outer layer of  Earth's crust" to "a collection of natural bodies on the surface of th

What are the abnormalities of gaze, What are the Abnormalities of gaze ...

What are the Abnormalities of gaze Normal gaze is when visual axes both eyes are parallel in primary gaze. when visual axes are not parallel in primary gaze, it is abnormal ga

Explain beginning and the end of the third interphase period, Q. What are t...

Q. What are the events that mark the beginning and the end of the third interphase period? What happens in the cell in this period? The third interphase period is the G2, It be

Explain precautions for detection of metanil yellow, Explain Precautions fo...

Explain Precautions for Detection of Metanil Yellow? 1. One should be careful while using concentrated HCl otherwise it may result in burn. 2. Amount of concentrated HCl in

Explain about ia muscle-spindle, Which of the following occur in response t...

Which of the following occur in response to an increase in the length of the right knee extensors in response to a quick tap applied to the right patellar tendon?  An increase in t

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