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

Biostatistic.., biostatistical application in biotechnology

biostatistical application in biotechnology

What are the vessels that carry blood to the kidneys, Q. What are the vesse...

Q. What are the vessels that carry blood to the kidneys? Is this blood venous or arterial? The renal arteries are ramifications of the aorta and so the blood filtered by the ki

The cell membrane or plasma membrane ruptures, What happens when the cell m...

What happens when the cell membrane or plasma membrane ruptures or breaks down? When cell membrane ruptures Ions leek out and if not repaired in time the cell will die. As we k

Define the functions of vitamin e, Define the Functions of Vitamin E? V...

Define the Functions of Vitamin E? Vitamin E is the major lipid-soluble antioxidant in the cell antioxidant defence system and is exclusively obtained from the diet. The main r

List a few shortcomings of native starches, List a few shortcomings of nati...

List a few shortcomings of native starches which make it unacceptable in certain food applications. Lack of free-flowing properties of water repellence of the starch granules,

Briefly explain about the chromium, Briefly Explain about the Chromium? ...

Briefly Explain about the Chromium? As you will go through this section, you will realize that compared to other minerals, the essentiality of chromium was recognized very late

Define ziehl-neelsen staining technique, Define Ziehl-Neelsen Staining Tech...

Define Ziehl-Neelsen Staining Technique? Ziehl-Neelsen staining is used for staining these bacteria. The technique was discovered first by Robert Koch during his pioneering inv

Biota of pelagic zone, Biota of Pelagic Zone Pelagic region constitute...

Biota of Pelagic Zone Pelagic region constitutes 90 per cent of the total ocean surface and is less rich in species and numbers of organisms than the two regions discussed bef

Female reproductive system - vagina, V AGIN A - Median, elastic, musc...

V AGIN A - Median, elastic, muscular tube 7.5 cm long. Open into vestibule by vaginal orffice. The lining forms vaginal rugae. Space between vaginal wall & cervix is forn

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