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

Pre-operative planning and implant exposure, Q. Pre-operative Planning and ...

Q. Pre-operative Planning and Implant Exposure? A hollow acrylic transitional restoration should be fabricated based on the original diagnostic preview with respect to the toot

Clinical features of gout disease, Q. Clinical Features of gout disease? ...

Q. Clinical Features of gout disease? You may be enjoying good heath but may suddenly get a severe attack. You wake up in the middle of the night, and your big toe feels as if

Genetics, What is pedigree analysis . How to solve the complicated question...

What is pedigree analysis . How to solve the complicated questions of pedigree . How to identify wheather it is dominant or reccessive its genotype

Describe the experimental approach using the tools, For many of the mammali...

For many of the mammalian Hox genes, it has been possible to determine that some of them are more similar to one of the insect HOM-C genes than to the others. Describe an experimen

What is spermatogenesis in human biology, What is Spermatogenesis in human ...

What is Spermatogenesis in human biology? The male sex cells, or sperm, are produced in the oval-shaped testes, the primary sex organs of the male reproductive system, in a pro

Phylum patehelminthes, explian te topic

explian te topic

Embryology in relation to taxonomy?, Embryology in relation to taxonomy

Embryology in relation to taxonomy?

Explain antigens, Explain Antigens ? A blood type, or group, is based ...

Explain Antigens ? A blood type, or group, is based upon the presence or absence of certain substances called antigens on the surface of the red blood cells. Antigens are subs

Define absorption, Define Absorption, transport and Excretion of phosphorus...

Define Absorption, transport and Excretion of phosphorus? As you have seen that food contains both organic and inorganic phosphorus, but most of it is absorbed in its inorganic

Culture negative endocarditis, In patients who have not received prior anti...

In patients who have not received prior antibiotics and who will ultimately have blood culture positive endocarditis, it is likely that 95 - 100 per cent of all cultures obtained w

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