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

Determine the central thermoreceptor neurons, Which of the following serves...

Which of the following serves as a sensor, or as part of a sensor, that functions in a negative feedback system? A. Central thermoreceptor neurons whose peripheral terminals ar

Define interaction of folate with vitamin c, Define Interaction of Folate w...

Define Interaction of Folate with Vitamin C? Vitamin C: Anaemia is observed in vitamin C deficient patients. Normochromic, normocytic or macrocytic or megaloblastic ana

Explain unacceptable aesthetics, Unacceptable Aesthetics The implant wh...

Unacceptable Aesthetics The implant which has successfully integrated with bone may still be a failure if the Prosthesis which it is designed to support does not meet with the

Protozoa, advantage sand disadvantages of protozoa

advantage sand disadvantages of protozoa

Food pyramid concept, Dietary counseling based on the food pyramid concept ...

Dietary counseling based on the food pyramid concept and the food to target concept will be quite useful in the management of the disease. The optimal age for discontinuing the

Describe surgical treatment prosthetic valve endocarditis, Describe Surgica...

Describe Surgical Treatment prosthetic valve endocarditis ? If there is definite indication for early surgery, the current understanding is to proceed with valve replacement ir

How many are the known amino acids, How many are the known amino acids that...

How many are the known amino acids that form proteins in living beings? There are twenty dissimilar known amino acids that form proteins related to the genetic code of the livi

Define the term - dna polymerase, Which of the following best describes the...

Which of the following best describes the reasons why the sliding clamp is able to confer processivity on DNA polymerase and not DNA primase? A. The sliding clamp coats the tem

Describe about the lysosomes enzymes, Describe about the Lysosomes enzymes ...

Describe about the Lysosomes enzymes Within the cytoplasm of the cell, several hydrolytic enzymes (lytic enzymes) capable of hydrolysing proteins, nucleic acids and carbohydra

Describe Fatty acids, In chemistry and particularly in biochemistry the fat...

In chemistry and particularly in biochemistry the fatty acid is a carboxylic acid with a long aliphatic tail (chain) that is either saturated / unsaturated. Many obviously occurrin

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