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

What does s-shaped pattern of population growth show, What does S-shaped pa...

What does S-shaped pattern of population growth show? How is J-shaped pattern dissimilar from it and why?

Show estrogen level and the lh level in the menstrual cycle, Q. What is the...

Q. What is the relationship between the estrogen level and the LH level in the menstrual cycle? What is the function of LH in the menstrual cycle and when does its blood concentrat

Occlusal load affect osseointegration, Occlusal load affect Osseointegratio...

Occlusal load affect Osseointegration The timing and amount of overload are critical for osseointegration. Occlusal loading early, where the primary stability is low leads to h

What are the main parts of the human ear, What are the main parts of the hu...

What are the main parts of the human ear? The human ear is separated into three mains parts: the external ear, the middle ear and the internal ear. Hearing Review - Image D

Zoology, main external morphological feature that differentiates platyhelmi...

main external morphological feature that differentiates platyhelminthes from nematodes

Excretory organs, what is the excretory organs of,rat,fish,lizard,frog,cock...

what is the excretory organs of,rat,fish,lizard,frog,cockroach,bird and snail.

Define rheology and syneresis, Define Rheology and syneresis Rheology:...

Define Rheology and syneresis Rheology:   the science of the deformation and flow of matter. It is the branch of physics concerned with the flow and change of shape of matter,

What is a prion, Q. What is a prion? The prion is an infectious (transm...

Q. What is a prion? The prion is an infectious (transmissible) protein able to replicate by transforming other proteins into a copy of the prion. The mechanism of the copying i

Explain about round worms, Which one of the following statements about cert...

Which one of the following statements about certain given animals is correct? 1. Round worms (Aschelminthes) are pseudocoelomates 2. Molluscs are acoelomates 3. Insects ar

Are gametes always made by meiosis, Q. Are gametes always made by meiosis? ...

Q. Are gametes always made by meiosis? In the haplontic haplobiontic life cycle and in the plant life cycle (diplobiontic life cycle) gametes are made by mitosis and not by mei

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