User Account

All Pages

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

The importance of buffer systems in the human body, Outline the importance ...

Outline the importance of buffer systems in the human body. In addition, describe the main biological buffer systems and how they function.

Name the gas which is used to regulate activity of the heart, In the more h...

In the more highly developed animals, like humans this gas is used to regulate activity of the heart, blood vessels and respiratory system. WORKING MUSCLES PRODUCE A LARGE AMOUNT O

#title.respiratio., i want notes for respiration in humans full qand to the...

i want notes for respiration in humans full qand to the point si i can learn them

What is guided bone regeneration, What is Guided bone regeneration Gui...

What is Guided bone regeneration Guided bone regeneration (GBR) allow the practitioner to place implants in patients who would not have received this treatment option a few y

Explain about chlorophylls - natural pigments, Explain about Chlorophylls ...

Explain about Chlorophylls Chlorophylls are used as colourants in a range of foodstuffs and both natural chlorophyll (containing magnesium as the central metal ion) and 'coppe

Fermentation, outline the process of yeast fermentation stating all enzymes...

outline the process of yeast fermentation stating all enzymes involved

Describe the developmental periods of coronary artery diseas, Describe the ...

Describe the Developmental Periods of coronary artery diseases? The development of coronary artery disease like many other diseases can be divided into the following periods:

Is acetylcholine used in consciousness, Q. Is Acetylcholine used in conscio...

Q. Is Acetylcholine used in consciousness? Cholinergic mechanisms are important in wakefulness and cortical activation. In general, increased acetylcholine is associated with w

Accident causation - accidents and losses, Accident Causation - Acciden...

Accident Causation - Accidents and Losses Several accidents occurs everyday year round in industries. We have already discussed different types of accidents and injuries t

Agro industrial-chelating agents, Chelating agents Oxalates: Only a ...

Chelating agents Oxalates: Only a few plants contain sufficient amounts of sodium and potassium oxalate to be considered toxic. Moreover, ruminants that consume these plants

Write Your Message!

Captcha
Order Assignment

Featured Services

Order Now

Popular Subjects

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!

Submit Assignment

Academics

Major Subjects

Majors

Get In Touch

whatsapp: +91-977-207-8620

Phone: +91-977-207-8620

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd