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

Ventricular septal defect might be cyanotic, A newborn baby with a patent f...

A newborn baby with a patent foramen ovale or a ventricular septal defect might be cyanotic (blue). Will a two-year-old with these defects also be cyanotic? Explain your answer.

Annelids - hormones in growth and reproduction, Annelids - Hormones in Grow...

Annelids - Hormones in Growth and Reproduction Studies on polychaetes have displayed that the endocrine glands play a key role in growth and reproduction. In addition to the b

Explain uristix benedicts test, Q. Explain Uristix Benedicts test? No s...

Q. Explain Uristix Benedicts test? No sugar/glucose is excreted in the urine in normal individuals. In diabetics, glucose is excreted by the kidneys when the blood sugar levels

Is there a respiratory pigment in the annelid blood, Is there a respiratory...

Is there a respiratory pigment in the annelid blood? The blood in beings of the phylum Annelida have the respiratory pigment hemoglobin (the similar found in chordates) and oth

Diversity in living organisms.., labelled diagram of male and female cockro...

labelled diagram of male and female cockroach with difference between them

Plant cells differ from animal cells, State the ways in which (a) all...

State the ways in which (a) all plant cells, (b) some plant cells vary from animal cells.    (a) All plant cells have a cellulose cell wall.  (b) Some plant c

Effect of smoking on body, EFFECT ON BODY - 1 .       CANCER - B...

EFFECT ON BODY - 1 .       CANCER - Benzpyrene present in tobacco is carcinogenic , responsible for lung cancer. Tobacoo chewing also produce oral cancer. Smoking m

SYMBIOTIC THEORY, WHAT IS SYMBIOTIC THEORY. PLEASE EXPLAIN THIS TO ME IN A ...

WHAT IS SYMBIOTIC THEORY. PLEASE EXPLAIN THIS TO ME IN A VERY SIMPLE WAY.

Carbon filtration or activated carbon process, Activated carbon has a much ...

Activated carbon has a much higher adsorption capacity than other materials. Carbon adsorbs objectionable odours, flavours, organic compounds and many hazardous chemicals. There

Analysis of the written reports, Analysis of the written reports   of  serv...

Analysis of the written reports   of  service in relation  to  the fitness, quality and efficiency of the service should be provided. The routine record-checking is always insuffic

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