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 tertiary level care - public nutrition, Define Tertiary level care -...

Define Tertiary level care - Public Nutrition? This is the highest level of health case available to the community for dealing with their most complex health problems, which ca

Will there be an opportunity to comment, Will there be an opportunity to co...

Will there be an opportunity to comment?  In a supplemental notice accompanying the last rule package, EPA is soliciting additional information and public comment on issues fro

Define factorial method to calculate nitrogen requirements, Define Factoria...

Define Factorial method to Calculate Nitrogen Requirements? The nitrogen requirements have been calculated by a factorial method suggested by different expert groups, described

Determine the method of recombination, Which of the following is a false st...

Which of the following is a false statement regarding the method of recombination or crossing-over? A. Crossing-over takes place during prophase I of meiosis B. Recombinatio

What is the average incubation period of chagas disease, Q. What is the ave...

Q. What is the average incubation period of Chagas' disease? What are some signs and symptoms of the acute phase of Chagas' disease? The Chagas' disease may or may not present

Gastrulation, GAS T RUL A TIO N - Morphogenic movements are occure...

GAS T RUL A TIO N - Morphogenic movements are occured. 3 germinal layers are formed. Ectoderm, endoderm & mesoderm. Endoderm is formed first. Mesoderm is formed in the

Hemoglobins - process of respiration, Hemoglobins - Process of Respiration ...

Hemoglobins - Process of Respiration Hemoglobins are the very well known of all respiratory pigments. The basic molecular unit of Hemoglobin contains a haem group bound to a

Define absorption, Define Absorption, Storage and Elimination of Pyridoxine...

Define Absorption, Storage and Elimination of Pyridoxine? Pyridoxine, pyridoxal and pyridoxamine (along with their phosphorylated forms) occur in plant and animal foods. The ph

Calf diptheria, Calf diptheria The disease is a serious one usually affect...

Calf diptheria The disease is a serious one usually affecting calves up to 2 years of age. The lesions are confined to larynx and pharynx, and consist of well-defined areas of nec

What are four initial stages of the embryonic development, Q. What are the ...

Q. What are the four initial stages of the embryonic development? The four early stages of the embryonic development are the blastula stage, the morula stage, the gastrula stag

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