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

Define the integrity of gut or colon - dietary fiber, Define the Integrity ...

Define the Integrity of gut or colon? Dietary fibre especially fermentable fibres play an important role in maintaining the integrity of gut. SCFAs generated during fermentatio

Plant, what is comretun

what is comretun

What is a pipette and how is it used, What is a pipette and how is it used?...

What is a pipette and how is it used? There are lots of types of pipettes (or pipette), but most are basically a fancier version of a medicine dropper or eye dropper. They are

Genetic or acquired deficiency of the vitamin k metabolism, Q. Why are some...

Q. Why are some kinds of hemorrhagic diseases caused by genetic or acquired deficiency of the vitamin K metabolism? Deficiency of vitamin K predisposes to hemorrhages since thi

Digestive system in the phylum arthropoda, Q. What is the kind of digestive...

Q. What is the kind of digestive system present in beings of the phylum Arthropoda? Are these animals deuterostomes or protostomes? The digestive tube of arthropods is complete

Explain the urinary system in human body, Explain the Urinary System in hum...

Explain the Urinary System in human body? The urinary system removes the waste materials produced as a result of all the metabolic process required to sustain life. The process

Determine the biological diversity of an ecosystem, Is monoculture a system...

Is monoculture a system that contributes to great biological diversity of an ecosystem? Monoculture means that in a large area a single crop (only single species of plant) is c

M - mode echocardiography, M-Mode was the earliest form of cardiac ultrasou...

M-Mode was the earliest form of cardiac ultrasound used clinically. It displays the amplitude signals of various structures along the length of ultrasound beam with distance or

What are worm infections, Q. What are worm infections? The Worm infecti...

Q. What are worm infections? The Worm infections are human diseases caused by nematodes(roundworms) or platyhelminthes (flatworms).

Cranium, need images of cranium

need images of cranium

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