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

Why is genetic homozygosity dangerous, Why is genetic homozygosity dangerou...

Why is genetic homozygosity dangerous to a nearly extinct species? Genetic homozygosity leaves no variation for natural selection to act on. Thus, a new disease could wipe

Determine factors affecting zn absorption - micro minerals, Determine Facto...

Determine Factors affecting Zn Absorption - Micro Minerals? In the last unit you have studied that absorption of various minerals (bioavailability) is influenced by number of f

What are risk factors for diseases, Q. What are risk factors for diseases? ...

Q. What are risk factors for diseases? The Risk factors for a disease are everything that contributes to increase the risk of the disease to appear. For example, for most cardi

Explain the process of determination of vitamin C, Determination of vitamin...

Determination of vitamin c Majority of chemical methods of determination is based on the rapid oxidation of ascorbic acid and therefore not too highly specific. Titration of

Non-poisonous snakes, Snakes Non-poisonous snakes can be brought into t...

Snakes Non-poisonous snakes can be brought into the classroom for observation. The diagram shows how a safe cage can be made for keeping a snake. The bottom should be covered w

Lentic ecosystems, Lentic Ecosystems Lakes are inland, depressions co...

Lentic Ecosystems Lakes are inland, depressions containing standing water. They vary considerably in area and depth. The largest lake in the world, the lake Superior in North

Nutrition, prove oxygen is produced during photosynthesis in the presence o...

prove oxygen is produced during photosynthesis in the presence of light

Factors affects oxygen dissociation curve -organic phosphate, Factors affec...

Factors affects Oxygen Dissociation Curve -Organic Phosphate The presence of organic phosphates in the red blood cells helps to explain many peculiarities of the oxygen dissoc

What is the role of the ozone layer for living beings, What is the role of ...

What is the role of the ozone layer for living beings? Ozone, O3, is a gas of the atmosphere that filters ultraviolet radiation from the sun disallowing most of that radiation

What do you mean by age pyramids, Q. What are age pyramids? Age pyramid...

Q. What are age pyramids? Age pyramids are the graphical representations in form of superposed rectangles each representing the number of individuals included in age ranges int

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