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

Saliva spreading - temperature relations in animals, Normal 0 f...

Normal 0 false false false EN-IN X-NONE X-NONE MicrosoftInternetExplorer4

Genotype for each individual, The allele that causes albinism (p) is recess...

The allele that causes albinism (p) is recessive to the allele for normal pigmentation (P). A normal woman whose father is an albino marries an albino man whose parents are both no

Leptospirosis, Leptospirosis Synonyms: Weil’ disease, canicola fever...

Leptospirosis Synonyms: Weil’ disease, canicola fever, 7-day fever Leptospirosis is an important and widespread zoonosis. It was first described by Adolf Weil in 1886 when h

According to their morphology how are bacteria classified, According to the...

According to their morphology how are bacteria classified? Bacteria present dissimilar morphological patterns. A bacterium can be classified into coccus, bacillus, vibrion or s

Determine the symptoms of campylobacteriosis, Determine the Symptoms of Cam...

Determine the Symptoms of Campylobacteriosis Symptoms:  C. jejuni infection causes diarrhoea, which may be watery or sticky and can contain blood and faecal  leukocytes (white

Explain vegetable sterilization process, Explain vegetable sterilization pr...

Explain vegetable sterilization process Canning, which involves heat sterilization, is one of the most important processes in vegetable preservation. As compared to other foods

What is the estimated percentage of water in the human body, Q. What is the...

Q. What is the estimated percentage (in mass) of water in the human body? Is this percentage expected to be larger in the adult or in the old individual? Approximately 65% of t

Bacillary dysentery and its etiology, Bacillary Dysentery   You have le...

Bacillary Dysentery   You have learnt about the diarrhoea in the foregoing sub-section, now let us take for example a child who has loose motion which contains blood and mucus

Explain about the adipose tissue - energy balance, Explain about the Adipos...

Explain about the Adipose tissue - energy balance? At this point, it will not be irrelevant to consider how exactly an increase in the fat depot takes place. For understanding

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