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

What is the single tooth implant case, In Single Tooth Implant Case: a)...

In Single Tooth Implant Case: a) Vertical distance available between the crest of residual ridge to the opposing occlusal surface. At least 7 mm space is required to a cementab

Preparation of nursing care plan, Practical Points in Preparation of Nursi...

Practical Points in Preparation of Nursing Care Plan You first review the nursing diagnosis, then assign this priority or the order in which these are to be met.  Alo

What is polyembryony, Q. What is polyembryony? Polyembryony is the phen...

Q. What is polyembryony? Polyembryony is the phenomenon in which a single embryo in its initial embryonic stage divides itself forming many new individuals of genetically ident

What is karyotype, What is karyotype? Name the karyotype is given to th...

What is karyotype? Name the karyotype is given to the set of chromosomes of an individual, generally when visualized and identified under the microscope. The visualization in g

Define the calcium toxicity, Define the Calcium Toxicity? Elevated bloo...

Define the Calcium Toxicity? Elevated blood calcium can occur in association with high parathyroid hormone, hyper- or hypothyroid conditions, bone metastasis, vitamin D toxicit

Explain about sex chromosomes, What is the other name given to sex chromos...

What is the other name given to sex chromosomes? Sex chromosomes are also known as allosomes (the other chromosomes that are not sex chromosomes are known as autosomes).

Pyramid of biomass - ecological pyramids, Pyramid of Biomass - Ecological P...

Pyramid of Biomass - Ecological Pyramids The pyramid of numbers is not a very good method to use if the organisms at different trophic levels are of greatly differing sizes. T

What does acute disease mean, What does acute disease mean? An sharp di...

What does acute disease mean? An sharp disease is which does not last for a longer duration n has no weakness in the body

How is price mechanism or supply and demand concerned, How is price mechani...

How is price mechanism or supply and demand concerned?   The price mechanism or supply and demand: The price mechanism or supply and demand is concerned with how buyers

Dietary recommendations during diarrhoea, Q. Dietary Recommendations during...

Q. Dietary Recommendations during Diarrhoea? The diet should take into account the normal RDI and various adjustments made to the quantity and quality of the foods to be given.

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