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

Is protein collagen has a high proportion of glycine, The protein collagen ...

The protein collagen has a high proportion (20% or more) of A. asparagine and glutamine B. proline and hydroxyproline C. glycine D. both A and B E. both A and C F

Natural selection under different environments condition, In the absence of...

In the absence of any large scale envirorrmental change that is, in more or less uniform environments, populations often maintain a stable genetic constitution with respect to many

PROTOZOA., WHAT ARE THE ADVANTAGES & THE DISADVANTAGES OF PROTOZOA?

WHAT ARE THE ADVANTAGES & THE DISADVANTAGES OF PROTOZOA?

TRACHEAL RESPIRATION, WHAT ARE TRACHEOLES

WHAT ARE TRACHEOLES

Show ions used in electrical impulse transmission in neurons, Q. What are t...

Q. What are the two major ions that participate in the electrical impulse transmission in neurons? The two major ions that participate in the electrical impulse transmission in

Show common contraindications of the contraceptive pills, Q. What are the c...

Q. What are the common contraindications of the contraceptive pills? There are medical reports associating the use of contraceptive pills with vomiting, headaches, vertigo, nau

Genetic diversity of population, Genetic Diversity of Population The b...

Genetic Diversity of Population The biological diversity of animals, plants, and microorganisms is of fundamental importance to human survival. The term "gene resources" may b

Response to heavy metal stress, Response to Heavy Metal Stress Severa...

Response to Heavy Metal Stress Several heavy metals emanating from industrial mining and sewage disposal operations contaminate the environment. Cadmium is a common contamina

Properties of fatty acid, A fatty acid having of a hydrocarbon chain and a ...

A fatty acid having of a hydrocarbon chain and a terminal carboxylic acid group that is shown in the figure.  Most  fatty  acids  are  in  biology  have  an  even  number  of carbo

Explain trypsin, Trypsin Trypsin is secreted in the inactive  form  try...

Trypsin Trypsin is secreted in the inactive  form  trypsinor  -  which is converted into the active form  trypsin by  the enzyme enterokinase secrated by  the duodenal mucosa.

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