cloning protocol, Biology

Assignment Help:
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. (10 Marks)

(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?) (10 Marks)

Related Discussions:- cloning protocol

Define decreased oxygen availability at high altitude, Define Decreased oxy...

Define Decreased oxygen availability at high altitude (Hypobaric hypoxia)? The governing biophysical factor at high altitude is decrease in barometric pressure with increase in

Draw a class diagram, Draw a class diagram: You are required to provid...

Draw a class diagram: You are required to provide a practical implementation in C++ for the following class diagram. You have to provide the solution in the form of classes in

Photometer-radiation instrument, Photometer Photometer (Figure shown be...

Photometer Photometer (Figure shown below) measures the intensity of light. The metallic plate A is a photocell which emits electrons from its surface when light of sufficient

Explain false left ventricular aneurysm, Explain False Left Ventricular Ane...

Explain False Left Ventricular Aneurysm? False Left Ventricular Aneurysm :  False aneurysm develops after acute rupture of an infarct. It is usually fatal, but a few survive

Tools of lower paieolithic age, Tools of lower paieolithic age Pleistoc...

Tools of lower paieolithic age Pleistocene times there were huge herds of very large mammals. Bones of such large mammals were found associated with the human fossil finds and

Physiological adaptaion, what are the physiological adaptation shown my pre...

what are the physiological adaptation shown my prey as defence

What is the energy source used in active transport, Q. What is the energy s...

Q. What is the energy source used in active transport through biological membranes? The energy necessary for active transport against the concentration gradient of the transpor

Destruction of microorganisms, Q. Destruction of microorganisms? Ans. ...

Q. Destruction of microorganisms? Ans. Destruction or control of growth of microorganisms is the basis of food preservation. To 'preserve' actually means fro keep safe, r

What is lipid-lowering trial, Q. What is Lipid-lowering Trial? Aggressi...

Q. What is Lipid-lowering Trial? Aggressive lipid-lowering drug treatment of persons at various risk levels reduces Coronary Artery Disease morbidity and mortality rates and in

Diffrence between fish epidermis and amphibian epidermis, Q. How different ...

Q. How different is the fish epidermis from the amphibian epidermis? The fish epidermis is very contains and thin mucus-secreting cells the fish skin doesn't present keratin, t

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