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

Why are the plants having single-seeded fruits, Why are the plants having s...

Why are the plants having single-seeded fruits and plants having fruits with surplus one seed? The Plants that produce single-seeded fruits, for instance, avocado and mango oft

Assessment of diastdlic function, Q. Assessment of Diastdlic Function? ...

Q. Assessment of Diastdlic Function? Myocardial ischemia Clters diastolic function of left ventricle. The earliest abnormality to appear with prolonged ischemia is delayed my

Physical weathering-weathering of rocks, Physical Weathering Mechanical...

Physical Weathering Mechanical forces acting upon the rocks cause physical weathering. Temperature fluctuations cause expansion and contraction of rock surface resulting in the

How do cam plants vary from both c3 and c4 plants, How do CAM plants vary f...

How do CAM plants vary from both C 3 and C 4 plants? CAM plants open their stomata at night, while C 3 and C 4 plants open their stomata during the day.

Explain about ia muscle-spindle, Which of the following occur in response t...

Which of the following occur in response to an increase in the length of the right knee extensors in response to a quick tap applied to the right patellar tendon?  An increase in t

Explain fats requirements during thyphoid, Explain fats requirements during...

Explain fats requirements during thyphoid Fats: Use of fats should be in moderation. This is because the typhoid patient has a compromised ability to digest and assimilate due

What is the meaning of trocophore, What is the meaning of Trocophore? T...

What is the meaning of Trocophore? This free-swimming ciliated larval stage is found in numerous animal phyla including Mollusca and Annelida. Larvae has a unique circle of pre

Control of radio-pollution , (i)        Area uses or perm...

(i)        Area uses or permit exposure to radiation should be Marked' Restricted Area 'or Radiation zone etc. (ii)        Avoid x-rays of chest and lower back in routine physic

Describe aerobic respiration to the pyruvic acid molecules, Q. What happens...

Q. What happens during aerobic respiration to the pyruvic acid molecules made by glycolysis? and What is the sequence of reactions that then follows? The pyruvic acid molecules

Primary tissues, what do primary tissues of a plant do

what do primary tissues of a plant do

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