cloning protocol, Sally Subcloner wants to clone a fragment into pUC19 (Map above

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)

Write Your Message!

Name

Email id

Message

Verfication Code

User Account

All Pages

cloning protocol, Biology

Related Discussions:- cloning protocol

Identify the abnormal protein, Identify the abnormal protein and state how ...

Explain functional property of emulsification, Explain Functional property ...

Heart in amphibians, Amphibia n - 3 chambered heart 2 auricles and ...

Phylum mollusca, what is the correct classification of phylum mollasca

How do organisms reproduce, Define sexual reproduction

How old is the earth, How old is the earth? It is believed that the ear...

History of microbiology, give me assignment plz

How can you describe the homeostasis, Q. What is the homeostasis? What are ...

Explain the radiographic examination of prothesis, Radiographic Examination...

Metazoan branches, Metazoan Branches The Kingdom Animalia comprises al...

Write Your Message!

Featured Services

Online Tutoring

Project Development

Exam Preparation

Course Help

Assignment Help

Popular Subjects

Assured A++ Grade

Academics

Major Subjects

Majors

Get In Touch

TERMS & POLICIES

HELP & SUPPORT