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

Does the fish heart pump venous or arterial blood, Does the fish heart pump...

Does the fish heart pump venous or arterial blood? The venous blood coming from the tissues enters the atrium and passes to the ventricle that then pumps the blood towards the

What is reduction, What is reduction? The most commonly understood defi...

What is reduction? The most commonly understood definition of reduction is the removal of oxygen or addition of hydrogen ions. However, chemically, reduction can be defined as

Embryology, Explain the gradient theoryof experimental embryology

Explain the gradient theoryof experimental embryology

Define the concept of health - public nutrition, Define the Concept of Heal...

Define the Concept of Health - Public Nutrition? The most widely accepted definition of health is the one given by WHO (1948) in the preamble to its constitution, Box 1 gives t

Class of subphylum uniramia - symphyla, Class of Subphylum Uniramia - Symph...

Class of Subphylum Uniramia - Symphyla Symphyla is yet other small myriapodous group that includes around 160 described species. These are also soil living forms and live in l

Determines the concentration of cortisol in the blood plasma, If one determ...

If one determines the concentration of cortisol in the blood plasma to be 10 micrograms/mL and that in a 24 hour period a person excretes 10 mgs of cortisol, calculate the volume o

Transplantation - clonal propagation, Transplantation - Clonal Propagation ...

Transplantation - Clonal Propagation The shoots or plantlets multiplied on a medium containing organic nutrients, show poor photosynthetic capability. Moreover, in these pla

What is the other name given to the sex chromosomes, Q What is the other na...

Q What is the other name given to the sex chromosomes? What is the function of the sex chromosomes? Sex chromosomes are also known as allosomes the other chromosomes that are n

Define limitations of agar syringe or agar sausage method, Define Limitatio...

Define Limitations of Agar Syringe or Agar Sausage Method? (i) It is applicable to surfaces with low contaminants. (ii) Problem of spreading colonies may occur. (iii) Col

Define nutrition support in postoperative nutritional care, Define Nutritio...

Define Nutrition Support in Postoperative Nutritional Care? Aggressive nutritional support is important to avoid postoperative complications and mortality. Routine postoperativ

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