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

Bacterial artificial chromosome (bac), Bacterial artificial chromosome (BAC...

Bacterial artificial chromosome (BAC) is a chromosome-like structure, made by genetic engineering. BAC is a cloning vector able to carrying between 100 and 300 kilo bases of targe

Procedure for use microbiological laboratory equipment, Define Procedure fo...

Define Procedure for use Microbiological Laboratory Equipments? Now carry out this activity following the steps given herewith: 1. Observe each instrument (as mentioned abov

Types of community, Types of Community On the basis of size and degree ...

Types of Community On the basis of size and degree of relative independence communities may be divided into two types: i) Major Community: These are large-sized, well org

Peroxisome and lysosome, e use itwhat is difference between peroxisme and l...

e use itwhat is difference between peroxisme and lysosome what is function of it and where w

What are colonies and societies, Q. What are colonies and societies? Th...

Q. What are colonies and societies? The Colonies are functional integrated aggregates formed by individuals of the similar species. The Colonies are often confused with a singl

Cellular Respiration, what two gases are released by animals during the pro...

what two gases are released by animals during the process of cellular respiration

Fertilization - development biology, Fertilization - Development Biology ...

Fertilization - Development Biology Previously you knew the process which leads to the differentiation of the male and female germ cells, the sperm and ova, respectively. In t

Reproduction, what is the arrangement of microtubules in the sperm

what is the arrangement of microtubules in the sperm

#title.ehr, Why have biologists and bioinformaticians embraced the Web as a...

Why have biologists and bioinformaticians embraced the Web as a vehicle for disseminating data so quickly, whereas clinicians and clinical informaticians have been more hesitant to

Explain galactose metabolism, Galactose Metabolism Galactose  goes to t...

Galactose Metabolism Galactose  goes to the liver via portal blood and is phosphorylated by galactokinase togalactose-  1  -phosphate (GIP) using ATP as  a phosphate donor as s

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