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

The skin, what is vaso-constriction

what is vaso-constriction

Define rhizoids - types of hyphae, Define Rhizoids - Types of Hyphae? ...

Define Rhizoids - Types of Hyphae? Rhizoids - These are brown slender root like structures which arise in cluster from each node of the stolon. These penetrate the substratum

Differences between anastral and astral mitosis, Q. What are the difference...

Q. What are the differences between anastral and astral mitosis? Astral mitosis is that in which there is formation of the aster a structure made by the centrioles. Anastral mi

Determine requirement of trace elements for infant, Determine Requirement o...

Determine Requirement of Trace elements for Infant? Most elements, such as zinc and iodine, hold a major significance in infant nutrition. Zinc is associated with growth while

Do we know what causes mental illnesses or how to treat them, Do we know wh...

Do we know what causes mental illnesses, or how best to treat them? Mental illnesses take various forms: debilitating sadness in depression; uncontrollable repetitive actions i

What are some examples of interspecific competition, Q. What are some examp...

Q. What are some examples of interspecific competition? Instance of interspecific competition are the dispute among worms, vultures, flies and microorganisms for carrion and th

Determine the major demographic processes, Determine the major demographic ...

Determine the major demographic processes? There are five major demographic processes, which are continually at work within the population namely, fertility, mortality, marriag

Histopathology, Liver necrosis?

Liver necrosis?

Cardiac care on admission for operation, Cardiac Care on Admission (First t...

Cardiac Care on Admission (First two hours) ECG is monitored by more than one lead (three to five). Left atrial pressure, arterial BP, central venous pressure, respiration

Which sugars does not contains an o-glycosidic bond, Which of the following...

Which of the following sugars does NOT contains an O-glycosidic bond? Select one: a. amilose b. amylopectin c. glycogen d. cellulose

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