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

Integumentary system - glands, Glands - 1 .      SEBACEOUS GLAND - ...

Glands - 1 .      SEBACEOUS GLAND - Absent in palm and sole. Holocrine in nature. Branched, alveoli are present, sac like in appearance. Generally attached to fo

What is congenital heart operations and interventions, What is Congenital H...

What is Congenital Heart Operations And Interventions Aftercare of the children who have undergone cardiac procedures and surgeries is as important, and in some occasions, more

Explain in brief about the cell membrane, Explain in brief about the Cell M...

Explain in brief about the Cell Membrane All cells, whether prokaryotic or eukaryotic, are bound by a limiting membrane called the cell membrane or the plasma membrane. The ce

Factors associated with accident, Factors Associated With Accident Co...

Factors Associated With Accident Cost in Administrative Functions Loss of prestige. Costs for notification, investigation and legal action. Loss of pu

Pennetula, identifying characters of pennetula

identifying characters of pennetula

Chordates, what are the general characters of chordates? At least 10 charac...

what are the general characters of chordates? At least 10 characters.

Steps for prevention industrial accidents, Steps for Prevention Industrial ...

Steps for Prevention Industrial Accidents These steps are as follow: Substitute a less hazardous equivalent. Remove worker from hazard (e.g. automate the sys

Find out the electric potential, Find the electric potential, taking zero a...

Find the electric potential, taking zero at infinity, at the upper right corner (the corner without a charge) of the rectangle in the figure. (Let y = 3.7 cm and x = 6.7 cm.)

Which are the plant tissues that cover the stem and leaves, Which are the p...

Which are the plant tissues that cover the stem and the leaves? The stem may be enclosed by epidermis (having stomata, cuticle and photosynthetic cells) as in monocots or, alte

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