User Account

All Pages

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

Describe the part of the female reproductive system, Q. What is the part of...

Q. What is the part of the female reproductive system where fecundation occurs? Fecundation in general occurs in the Fallopian tubes but it can also take place within the uteru

Crucial periods of a woman''s life - pregnancy and lactation, Define crucia...

Define crucial periods of a woman's life - pregnancy and lactation? In this unit, we studied about the most crucial periods of a woman's life, pregnancy and lactation, especial

Define requirements of fat in postoperative nutritional care, Define Requir...

Define Requirements of Fat in Postoperative Nutritional Care Adequate amount of fat is needed to build up and maintain tissue fat reserves. Depending upon the existing health a

Requirements of various nutrients in constipation, Q. Requirements of vario...

Q. Requirements of various nutrients in constipation? The requirements of various nutrients are not altered in constipation. It is essentially a normal balanced diet (normal RD

Determine the concentration of your original dna sample, For many years, st...

For many years, students who have occupied my Molecular Biology course have now applied for lab tech jobs exact after graduation. But I have NEVER had a student fail to obtain a jo

Inferior epigastric artery-other arterial conduits, Inferior Epigastric Art...

Inferior Epigastric Artery (I.) :  This is a branch of external iliac artery supplying the abdominal wall. It is raised as a free graft for CABG. The usual length availab

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,

Dietary modification, Q. Dietary modification? Dietary modification inc...

Q. Dietary modification? Dietary modification includes nutritional support that allows normal growth and development. Plasma phenylalanine concentration between the range of 40

Define vapor-pressure lowering and osmotic pressure, Define Vapor-pressure ...

Define Vapor-pressure lowering and Osmotic pressure? 1. Vapor-pressure lowering: the decrease in the vapor pressure of a solution containing nonvolatile solutes, compared to th

Determine the symptoms of cholera, Determine the Symptoms of Cholera  A...

Determine the Symptoms of Cholera  An infective dose of around one million organisms should be ingested to cause illness. The bacteria adhere to the small intestine wall, where

Write Your Message!

Captcha
Order Assignment

Featured Services

Order Now

Popular Subjects

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!

Submit Assignment

Academics

Major Subjects

Majors

Get In Touch

whatsapp: +91-977-207-8620

Phone: +91-977-207-8620

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd