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

Determine the fransworth 100 hue panel test, Determine the Fransworth 100 H...

Determine the Fransworth 100 Hue Panel Test This is a simple and useful test using colour chips arranged in a particular sequence. Colour deficient patients make errors in arra

Explain acute renal failure, Explain Acute Renal failure Acute Renal f...

Explain Acute Renal failure Acute Renal failure:  Renal failure associated with burns or other trauma or with sharp infection or obstruction of the urinary tract.

Treatment and nursing management of cleft lip, Treatment and Nursing Manage...

Treatment and Nursing Management The initial treatment is surgical repair of  the cleft. The timing of the repair varies from surgeon to surgeon, but most prefer cleft lip rep

Explain the completed test - most probable number test, Explain the Complet...

Explain the Completed Test - Most Probable Number Test? Coliform colonies on EMB or Endo agar are further examined by completed test by inoculating lactose broth and nutrient a

Explain about conjugated proteins, Conjugated Proteins Conjugated prote...

Conjugated Proteins Conjugated proteins are composed of easy proteins combined with a non- proteinous substance.  The non-proteinous substance is known as prosthetic group o

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

Does the trend of the change in shape of the graph, Does the trend of the c...

Does the trend of the change in shape of the graph as temperature increases differ when a different gas is examined?

Alimentory canal, how to do assignment on alimentory canal

how to do assignment on alimentory canal

What do you mean by alzheimer''s disease, Q. What is Alzheimer's disease? ...

Q. What is Alzheimer's disease? The Alzheimer's disease is a degenerative disease of the central nervous system in which the patient has progressive dementia and alteration of

What do you mean by fixation?, What do we mean by fixation? Fixation is...

What do we mean by fixation? Fixation is the process of preserving internal and external structures of microorganisms.  It leads to the killing of microbial cells and their fir

Write Your Message!

Captcha
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!

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd