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

How is the fluid that fills the nucleus called, The aqueous fluid that fill...

The aqueous fluid that fills the nuclear region is known as karyolymph, or nucleoplasm. In the fluid there are proteins, enzymes and other significant substances for the nuclear me

How did pasteur experiment vary from spallanzani, How did Pasteur's experim...

How did Pasteur's experiment vary from Spallanzani's experiment? Instead of sealing the flask in the experimental group after boiling, Pasteur used a flask with a curved neck,

Succession - community change, Succession - Community Change The facto...

Succession - Community Change The factors like fire, floods and human interventions affect an ecosystem considerably. They often lead to the depletion or stripping off of orig

Barrier function of epithelium and endothelium in corneal, Explain about th...

Explain about the barrier function of epithelium and endothelium in corneal hydration. Barrier Function of Epithelium and Endothelium: Epithelium offers twice the resista

Illustrate the methods to evaluate osseointegration, Q. Illustrate the Meth...

Q. Illustrate the Methods to evaluate osseointegration? The criteria of success in implant dentistry remains complex. The vast majority of clinical reports noting success and f

Define interaction of niacin with other nutrients, Define Interaction of ...

Define Interaction of niacin with other Nutrients? You may recall reading earlier that trypotophan present in dietary proteins is converted to niacin. There is an interdepend

How to produce a similar sickling effect, Which of the following amino acid...

Which of the following amino acids would you expect to produce a similar sickling effect if placed at position 6? Select all that apply. 1. Arginine 2. Leucine 3. Lysine

Palynofossils (microfossils), Palynofossil s (Microfossils) - Fossils ...

Palynofossil s (Microfossils) - Fossils of planktons, diatoms, unicellular algae (diatoms) and shelled protozoans. (Heliozoans, Radiolarian and Foraminifera). Its finding in s

Deficiency diseases-post-parturient haemoglobinuria (pph), Post-parturient ...

Post-parturient haemoglobinuria (pph) in cattle and buffaloes The post-parturient haemoglobinuria (PPH) is an acute disease of high yielding buffaloes and cows. The disease oc

Determine about the multicellular organisms, Another possible way to classi...

Another possible way to classify organisms would be to separate them into unicellular and multicellular organisms. Explain why this is not a useful classification system. Other

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