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

Pressure and continuity, Pressure and Continuity Pressure Water...

Pressure and Continuity Pressure Water pressure increases with depth which varies in the ocean from 1 atmosphere where at the surface to 1000 atmosphere at the greatest

What are the enzyme cofactors, Q. What are the enzyme cofactors? Some o...

Q. What are the enzyme cofactors? Some of enzymes require other associated molecules to work. These molecules are known as enzyme cofactors and they can be, for example, organi

Which are the brain regions associated with memory, Which are the brain reg...

Which are the brain regions associated with memory? According to researchers some of the main regions of the nervous system associated with the memory phenomenon are the hippoc

What is hazard identification, What is Hazard Identification Hazard  I...

What is Hazard Identification Hazard  Identification :  The  identification  of  known  or potential  health effects associated with  a  certain  agent.

Why mosquito bites and it causes itching, Why mosquito bites and it causes ...

Why mosquito bites and it causes itching? A mosquito does not really bite you, of course. It sucks your blood. To help enable effective blood sucking, it firstly injects ant

What is the structural flat representation of an amino acid, What is the st...

What is the structural flat representation of an amino acid molecule? An amino acid has a central carbon to which a carboxyl group binds on a side and to which a -R (variable r

Explain aerobic cellular respiration during muscle exercise, What happens w...

What happens when the oxygen supply is insufficient to maintain aerobic cellular respiration during muscle exercise? If oxygen from hemoglobin or myoglobin is not enough for th

Cells, Cell theory exception

Cell theory exception

Buffalo-pox, B u f fa l o- p o x The disease i...

B u f fa l o- p o x The disease is caused by an orthopox virus, closely related to the vaccinia virus. It is not clear whether it should be considered

What is hemodialysis, What is hemodialysis? Hemodialysis is the artific...

What is hemodialysis? Hemodialysis is the artificial blood filtration made by exact machines in substitution of the kidneys. Hemodialysis may be essential in patients suffering

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