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

Explain various types of activities of a cell, The main arena of various ty...

The main arena of various types of activities of a cell is: 1. Plasma membrane 2. Mitochondrian 3. cytoplasm 4. Nucleus Cytoplasm

What is the life cycle type of bryophytes, Q. What is the life cycle type o...

Q. What is the life cycle type of bryophytes? As in all plants the life cycle of bryophytes is diplobiontic (alternation of generations) and in bryophytes the lasting form is t

Preparation of the laboratory samples, Q. Preparation of the Laboratory Sam...

Q. Preparation of the Laboratory Samples From the primary sample while selecting a representative laboratory sample, we need to consider the following: a) Laboratory sampl

Monkeey Opsin Evolution, Why does having three color receptors (a.k.a. opsi...

Why does having three color receptors (a.k.a. opsins) lead to a more complex color perception than just two?

Endothelium derived constricting factor, Endothelium is a source of vasocon...

Endothelium is a source of vasoconstrictor factor also. The best characterized of these are the endothelins. Endothelium produces only ET1. Unlike NO, which is released rapidly in

Explain why this drug would eventualy kill a person, Dinitrophenol, DNP, wa...

Dinitrophenol, DNP, was prescribed in low doses in the 1940's to help obese patients lose weight. It's use for this purpose was discontinued when several users died. DNP makes the

Micro, What are the differences between the two domain

What are the differences between the two domain

Phylum protozoa, #question.sir please explain amoeba as in example of proto...

#question.sir please explain amoeba as in example of protozoa

Explain foscarnet, Foscarnet  (Foscavir)  Foscarnet is an alternative t...

Foscarnet  (Foscavir)  Foscarnet is an alternative to ganciclovir and valganciclovir for treatment of CMV infection. It is approved for use in CMV retinitis, including progress

How to identify the metal out of which the wire is made, A wire 50.0 m long...

A wire 50.0 m long and 2.00 mm in diameter is connected to a source with a potential difference of 9.11 V, and the current is found to be 33.7 A. Assume a temperature of 20°C and,

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