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

Nitrogen control of nitrogen assimilation, Nitrogen Control of Nitrogen Ass...

Nitrogen Control of Nitrogen Assimilation N 2 -fixer like Klebsiella pneumoniae and Nostoc can grow with N 2 , NO - 3 or NH + 4 as nitrogen source. You would like to know

Photosynthesis., C4 plants have higher net photosynthetic rate because ...

C4 plants have higher net photosynthetic rate because a.they have no photorespiration b.can photosynthesise in low in

What is iodoacetate inhibitor, What is Iodoacetate inhibitor Iodoacetat...

What is Iodoacetate inhibitor Iodoacetate is the inhibitor of glyceraldehyde-3-phosphate  dehydrogenase.

Describe valvar aortic stenosis murmurs, Describe Valvar Aortic Stenosis Mu...

Describe Valvar Aortic Stenosis Murmurs ? Characteristic: Harsh, rasping crescendo-decrescendo murmur best heard anywhere in a straight line from right 2nd ICS to apex ("sash"

What is bile salts, What is Bile salts Bile salts have a considerable...

What is Bile salts Bile salts have a considerable ability to lower surface tension. By  this property, they prevent  the coming together of the small fat droplets in  the  in

Radial and biradial - metazoa, Radial and Biradial-Metazoa Radial sym...

Radial and Biradial-Metazoa Radial symmetry is the symmetry in which the parts are so arranged around a central axis or shaft, like the spokes of a wheel, that any vertical c

Evaluation of osseointegration, Q. Evaluation of osseointegration? All ...

Q. Evaluation of osseointegration? All the parameters of proper implant selection, atraumatic sequential osteotomy preparation and the attainment of primary stability are focus

Mammalian lungs - respiration, Mammalian Lungs - Respiration In this w...

Mammalian Lungs - Respiration In this we will study mainly mammalian lungs as it is the best representative of a respiratory surface adapted for terrestrial respiration. For t

Define the term- new life for ancient dna, Based on your reading of the art...

Based on your reading of the article "New Life for Ancient DNA" which of the following is a  false statement in regards to the comparison among genes in extinct and living species?

Define objective of taxonomy -assemblage of knowledge gained, Define Object...

Define Objective of Taxonomy - Assemblage of Knowledge Gained? A second objective of taxonomy is the assemblage of knowledge gained. This is usually in the form of treatises us

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