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

Define the chemical fixation method, Define the Chemical Fixation Method? ...

Define the Chemical Fixation Method? Here chemical fixatives containing ethanol, acetic acid and formaldehyde etc. penetrates the cell and inactivates and immobilizes cellular

Mechanism of stomatal opening, Mechanism of Stomatal Opening It has be...

Mechanism of Stomatal Opening It has been known for over a century that stomata open because of reversible turgor changes in the guard cells. Stomata open when turgor in the g

What are the disadvantages of ridgemapping, Disadvantages of Ridgemapping ...

Disadvantages of Ridgemapping  a) Traumatic procedure b) Time consuming procedure and needs more armamentarium c)  There is an inherent risk of inaccuracy especially whil

Explain full liquid diet, Explain Full Liquid Diet Full Liquid Diet:  ...

Explain Full Liquid Diet Full Liquid Diet:   This diet provides foods and fluids that are liquid or semi liquid at room temperature. The type of food provided may vary dependi

What do you mean by borrowed materials, Q. What do you mean by Borrowed Mat...

Q. What do you mean by Borrowed Materials? For the study of as much relevant material as possible the material is obtained on loan. Borrowed specimens benefit the' lending inst

Gangrenous dermatitis (necrotic dermatitis), G a ngrenous dermatitis (nec...

G a ngrenous dermatitis (necrotic dermatitis) A bacterial condition in chickens caused by spores of Clostridium septicum , sometimes that of Cl. novyi / Cl. edematiens a

What is low - density lipoprotein receptor pathway, Q. What is low - densit...

Q. What is low - density lipoprotein receptor pathway? Ans. The increasing cellular free cholesterol generated regulates the activities of two enzymes that are of crucial

Describe the importance of neuropsychological tests, Describe the importanc...

Describe the importance of Neuropsychological tests Neuropsychological tests are generally thought of as assessment instrument for a variety of cognitive, perceptual, and motor

basic phases of pcr, What are three basic phases of PCR?  Which phase of t...

What are three basic phases of PCR?  Which phase of these three phases is used for analysis for the end-point PCR? Why? Which phase is used for analysis for the quantitative real-t

What is meant by binomial nomenclature, What is meant by binomial nomenclat...

What is meant by binomial nomenclature and what are its basic rules? Scientific nomenclature of a species must have, at least, two names: one that classifies it as genus and th

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