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 drugs, What is drugs? Intravenous access must be established. A...

What is drugs? Intravenous access must be established. Although administration of drugs through a central vein is ideal in a low cardiac output situation, it is rarely possible

Explain purpose of therapeutic dietary adaptations, Purpose  of  Therapeuti...

Purpose  of  Therapeutic Dietary Adaptations A therapeutic diet is a quantitative/ qualitative modified version of a basic nutritious diet which has been tailored to suit the c

What is gametogenesis, What is the type of cell division that allows sexual...

What is the type of cell division that allows sexual reproduction? What is gametogenesis? Meiosis is the type of cell division that permits sexual reproduction since it decreas

Determine the amount of glucose in the blood, During a routine annual physi...

During a routine annual physical,a patient was checked to determine the amount of glucose in the blood.After the assay,It was found that the glucose that the glucose concentration

Define the structure of moulds, The structure of moulds When the four p...

The structure of moulds When the four pure cultures of mould have reached a vigorous state of growth study each one with a hand lens. See if you can see stalks with tiny black

Skin, Two adaptation of the skin against attack by micro-organisms

Two adaptation of the skin against attack by micro-organisms

Factors affects oxygen dissociation curve - Carbon dioxide , Factors affect...

Factors affects oxygen dissociation curve - Carbon dioxide Carbon dioxide lowers the oxygen affinity of haemoglobin even if the pH is kept constant. This effect is due to the

Bacteria is an obligate anaerobe, Q. What is signifying when it is said tha...

Q. What is signifying when it is said that a bacteria is an obligate anaerobe? Obligate anaerobes are those living beings that do not survive in the presence of oxygen. For exa

Water absorption in digestive system of mammals and bird, Q. Compared to ma...

Q. Compared to mammals do birds absorb more or less water in their digestive system? Why is this phenomenon an adaptation to flight? Bird feces are more liquid than mammal a fe

Explain some physiological changes during pregnancy, Explain some Physiolog...

Explain some Physiological Changes during Pregnancy? A whole new life begins at conception. Organ systems develop rapidly and nutrition plays many supportive roles. Pregnancy f

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