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

Why is rubella during gestation a threat to the fetus, Why is rubella durin...

Why is rubella during gestation a threat to the fetus? If occurring during gestation rubella is a dangerous disease due to the virus crosses the placenta and contaminates the f

Excretory, what are the excretory organs protozoa

what are the excretory organs protozoa

Bladder worm, what is the function of the bladder in the bladder worm

what is the function of the bladder in the bladder worm

Local control mechanism within the circulatory system, Which of the followi...

Which of the following is true for active hyperemia, a local control mechanism within the circulatory system? A. There will be an increase in force developed by smooth muscles

Biological diversity protection, Q. How can a great biological diversity pr...

Q. How can a great biological diversity protect an ecosystem from environmental damage? Why are less biodiverse ecosystems at risk of suffering deep biological harm if submitted to

Why the recipient important in transfusions, Why is the determination of th...

Why is the determination of the blood types of the donor and of the recipient important in transfusions? Red blood cells have dissimilar antigens in the outer surface of their

Define that dna is the genetic material, How did Alfred Hershey and Martha ...

How did Alfred Hershey and Martha chase arrive at the conclusion that DNA is the Genetic material?

Nervous tissue and its components, NE R V OU S TISSUE The nervous...

NE R V OU S TISSUE The nervous tissue in general develops from the ectoderm of the embryo, but the microgliocytes. It does not have a matrix. The special properti

#title, WHAT IS THE FUNCTION OF CELL COAT

WHAT IS THE FUNCTION OF CELL COAT

Vesicular exanthema, Vesicular exanthema Vesicular exanthema of pigs close...

Vesicular exanthema Vesicular exanthema of pigs closely resembles foot-and-mouth disease in these animals. The virus causing the disease belongs to the genus Calicivirus in the fa

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