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

Pollenkitt - pollen wall, Pollenkitt - Pollen Wall The lipoidal layer ...

Pollenkitt - Pollen Wall The lipoidal layer found on the outside of the mature pollen grains of many insect pollinated species is known as the pollenkitt. In addition to lipid

What is the clitellum of earthtworms and where it is located, What is the c...

What is the clitellum of earthtworms and where it is located? The clitellum is a special region of the annelid constituted by rings (metameres) with reproductive function. It c

How vitamin e is directly related to selenium and pufa, How Vitamin E is di...

How Vitamin E is directly related to selenium and PUFA? Selenium: An inter-relationship exists between vitamin E and selenium, as selenium functions as an integral par1

What do numeric pyramids represent, What do numeric pyramids represent? ...

What do numeric pyramids represent? Numeric pyramids show the number of individuals in each trophic level of a food chain.

State the objectives of neuropsychological assessment, State the goals of N...

State the goals of Neuropsychological assessment Neuropsychological assessment therefore has twin goals. i) The first goal is to recognize the disrupted psychological compo

Extinction, EXTINCTIO N  - It refers to disappearance of a species from...

EXTINCTIO N  - It refers to disappearance of a species from earth when its last surviving member dies. Organic evolution not only creats new sps. but also eliminates some ol

Define maxillofacial prosthodontics, Q. Define Maxillofacial Prosthodontics...

Q. Define Maxillofacial Prosthodontics? Dental implants are now been increasingly used for Maxillofacial Prosthodontics. Maxillofacial Prosthodontics involves the prosthetic re

Explain inhibitors, Explain Inhibitors Inhibitors: Citrate synthase i...

Explain Inhibitors Inhibitors: Citrate synthase is inhibited by ATP, NADH, succinyl CoA and acyl CoA derivative of fatty acids (fatty acyl CoA). The rate of the reaction  is

What do numeric pyramids represent?, What do numeric pyramids represent? ...

What do numeric pyramids represent? The Numeric pyramids represent the number of individuals in each trophic level of a food chain.

Explain properties of amylopectin and amylose, Properties of Amylopectin an...

Properties of Amylopectin and Amylose In plants, starch is packaged in granules, which vary in their  shape and size, when isolated from different sources. The size of these gr

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