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

Cross contaimenation, how to prevent cross contamenation

how to prevent cross contamenation

How are the circulatory systems of animals classified, Q. How are the circu...

Q. How are the circulatory systems of animals classified? A circulatory system is classified as open or closed. In open circulatory systems blood gets out of vessels and flows

What are the factors that for influencing photosynthesis, Q. What are the f...

Q. What are the factors that for influencing photosynthesis also interfere with the gross primary productivity? Mostly light and water, but as well mineral salts, carbon dioxid

Health stroke scale, Health Stroke Scale: Stroke is a leading cause of...

Health Stroke Scale: Stroke is a leading cause of death, hospitalization, disability and health care expenditure in U.S. (Fonarow G. et al., 2012).  Stroke is a medical condit

Explain the genioglossus, Genioglossus Originates from superior genial ...

Genioglossus Originates from superior genial tubercles which are located near the crest of the alveolar ridge in atrophic mandible. Therefore, one should be aware of it during

What are intraspecific ecological interactions, What are intraspecific and ...

What are intraspecific and interspecific ecological interactions? Intraspecific ecological interactions are those among individuals of the same species. Interspecific ecologic

Ecology.., write the charecteristics of lake ecosystem

write the charecteristics of lake ecosystem

TISSUES, HOW TO WRITE A CONCLUSION ON A ASSIGNMENT OF EPITHELIAL TISSUES

HOW TO WRITE A CONCLUSION ON A ASSIGNMENT OF EPITHELIAL TISSUES

How might stem cells be used to treat brain diseases, How might stem cells ...

How might stem cells and other tools of "regenerative medicine" be used to treat brain diseases? Fuelled by the hope which stem cells may have the capacity to rebuild virtually

Objectives of taxonomy, Q. Objectives of taxonomy? Plants and animals b...

Q. Objectives of taxonomy? Plants and animals both have a great significance and unparalleled impact on human life. Most of our needs are fulfilled with plants such as foodstuf

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