Cloning protocol, Biology

Assignment Help:

1945_Alkaline Phosphate treatment.png

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.

(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?)


Related Discussions:- Cloning protocol

Fructose-1, Fructose-1,6-bisphosphate  and Fructose-6-phosphate : The  conv...

Fructose-1,6-bisphosphate  and Fructose-6-phosphate : The  conversion of  fructose-1,6- bisphosphate  to fructose-6-phosphate is  catalysed by fluctose-1,6-bisphosphatase  which  i

Define about the resistant starch, Define about the Resistant Starch? U...

Define about the Resistant Starch? Until 1980's starch was thought to be completely hydrolyzed and absorbed from the small intestine of man, independent of its source, type and

What are the cells that form the cartilaginous tissue, Q. What are the cell...

Q. What are the cells that form the cartilaginous tissue? The main cells of the cartilages are the chondrocytes originated from the chondroblasts that secrete the intersticial

Cyclosporine, Cyclosporine Immunosuppressive agent that is selective...

Cyclosporine Immunosuppressive agent that is selective for lymphocytes mainly "T" lymphocytes. Route: Oral, parental, dose as prescribed. Side-effects: Ne

Transport into the cytosol, Fatty  acids  are  synthesized  in  the  cytoso...

Fatty  acids  are  synthesized  in  the  cytosol,  but  acetyl  CoA  is  produce  from pyruvate in the mitochondria. Therefore the acetyl CoA must be transferred  from  the mitocho

Hypoglycaemic treatment, Hypoglycaemic coma is a medical emergency. A loss ...

Hypoglycaemic coma is a medical emergency. A loss of consciousness is due to low sugar level. Patient should immediately be given biscuits, chocolates, sweet, fruit. If unconscious

Absorption of glucose, Normal 0 false false false EN-IN...

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

Types of ecosystem, Ecosystems are so varied in form and structure that wha...

Ecosystems are so varied in form and structure that whatever has a distinct community of its own, can be called an ecosystem eg. A crop field, grass land, barks etc. all the ecosys

What is the etiological agent of visceral leishmaniasis, Q. What is the eti...

Q. What is the etiological agent of visceral leishmaniasis? How the disease transmitted and what is are its typical manifestations? The Visceral leishmaniasis is caused by the

hypotheses testing, Celia is a nurse in a geriatric ward.  She noticed tha...

Celia is a nurse in a geriatric ward.  She noticed that older persons in her care are having problems sleeping at night.  She decided to introduce non-pharmocologic ways of relaxat

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