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

Thikka desease of ground nut, Say about thikka desease of ground nut

Say about thikka desease of ground nut

Define discovery of other essential nutrients, Define Discovery of other Es...

Define Discovery of other Essential Nutrients? Following the identification of the food factors involved in beriberi, pellagra and scurvy, the search was on for other water-sol

Illustrate the techniques of neurology, Illustrate the techniques of neurol...

Illustrate the techniques of neurology A long-standing technique in neurology has been observed the effects on behaviour of lesions (cutting) or ablation (removal) of nerve tis

Blood collection, Blood Collection: Blood for analysis may be obtained...

Blood Collection: Blood for analysis may be obtained from veins, arteries, or capillaries. Venous blood is usually the specimen of choice, and venipuncture is the method for

Interrupted suture technique-prosthetic aortic valve, Interrupted suture te...

Interrupted suture technique: Synthetic braided 2 '0' sutures (ethibond) with 17mtn needles on either end and a pledget is used. Alternate green and white sutures are taken aft

Explain magnitude and direction of the electrostatic force, Three charges a...

Three charges are arranged as shown in the figure below. Find the magnitude and direction of the electrostatic force on the charge q = 4.78 nC at the origin. (Let r12 = 0.260 m.)

Why the inflammatory response is considered a body, List specific reasons w...

List specific reasons why the inflammatory response is considered a body defense mechanism.

Role of endarterectomy, Role of Endarterectomy :  Endarterectomy is done a...

Role of Endarterectomy :  Endarterectomy is done as a semi open technique. 5-10mm long incision is made on the outer layers of the coronary artery and plain developed in 11ic thic

Digestive system in cockroach, DIGESTIVE SYSTEM IN COCKROACH ALIMEN ...

DIGESTIVE SYSTEM IN COCKROACH ALIMEN T A R Y CANAL 6-8 cm. long. Coiled, present from mouth to anus. Differentiated into 3 parts -      1. Foregut            2. M

The emergency department due to the babys persistent, A mother has brought ...

A mother has brought her 2-week-old infant to the emergency department due to the baby's persistent and increasing jaundice. Blood testing reveals that the infant's unconjugated bi

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