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

Determine the case when starches treated with heat, Determine the case when...

Determine the case when starches treated with heat In the case of starches treated with heat in presence of acid or alkali, the alteration is a minor fragmentation; similarly,

Bachelor of nursing, case study

case study

Genetics, Ask question importance of genetics in human life #Minimum 100 w...

Ask question importance of genetics in human life #Minimum 100 words accepted#

Define conservation genetics and hybridization, Define Conservation genetic...

Define Conservation genetics and hybridization? Is the genetic integrity of European wolves threatened by hybridization along with free-ranging dogs? To ascertain the extent of

Problem with role of lymphatic system, Normal 0 false false ...

Normal 0 false false false EN-IN X-NONE X-NONE MicrosoftInternetExplorer4

What are some examples of parasitism, What are some examples of parasitism?...

What are some examples of parasitism? Classical instances are the parasites of humans (host), as the trypanosome that causes Chagas' disease, the HIV virus (AIDS), the bacteria

What volume of 4x protein sample buffer, What volume of 4x protein sample b...

What volume of 4x protein sample buffer do you need to add to 15 ml of a cell extract in order to load it on a gel?

Depleted (vulnerable) species, Depleted (Vulnerable) Species These are...

Depleted (Vulnerable) Species These are the species whose numbers are greatly reduced from those of the recent past, and they are continuing to decrease. It is the continued d

Sunlight used for sterilization, Q. Sunlight used for sterilization? It...

Q. Sunlight used for sterilization? It possesses appreciable bactericidal activity under natural condition. The action is primarily due to content of the ultraviolet rays; howe

What do you mean by punch cards, Q. What do you mean by Punch cards? Pu...

Q. What do you mean by Punch cards? Punch cards are used when the number of taxa to be keyed out is large. In this type of cards the holes at numbers corresponding to a charact

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