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

Explain disease motion sickness, Motion Sickness  The pathogenesis of m...

Motion Sickness  The pathogenesis of motion sickness is poorly under- stood. The prescription cholinergic blocker scopolamine in a patch or oral formulation can decrease sympto

Zoology, #question. what is cloning.

#question. what is cloning.

Describe the external features of the heart, Question 1 Describe the exter...

Question 1 Describe the external features of the heart. Add a note on circulation of blood within the heart Question 2 Discuss the major joints of thorax and pelvis Quest

What are sori, What are sori? The word "sori" is the plural form of "so...

What are sori? The word "sori" is the plural form of "sorus". In ferns, a sorus (pl. sori) is a cluster of sporangia on the edge or underside of a fertile frond. In lots of spe

Explain procedure for the use of light microscope, Explain Procedure for th...

Explain Procedure for the use of Light Microscope? Now carry out the exercise following the steps enumerated herewith. 1. Place the microscopic slide with any specimen on th

Explain about the musculoskeletal system, Explain about the Musculoskeletal...

Explain about the Musculoskeletal System? Osteopenia (decrease in bone mineral content) is observed with aging. There is an average of 30% decline in the bone mineral density f

Monocot and dicot plants, project formate for monocot and dicot plants in f...

project formate for monocot and dicot plants in feild per month

Explain common concerns during pregnancy, Explain Common Concerns during Pr...

Explain Common Concerns during Pregnancy? Many of the physiological changes that occur during pregnancy affect the digestive tract and may cause discomfort to the mother. Most

Can the heat capacity of water be taken small or large, Q. Can the heat cap...

Q. Can the heat capacity of water be taken small or large ? What is the biological importance of that characteristic? Ans. From Thermology it is define that the quantit

When a fatty acid reacts with thew glycerol, When a fatty acid reacts with ...

When a fatty acid reacts with glycerol, the result is- Select one: a. Formation of an amide b. Formation of an ester c. Formation of hydrocarbon d. Formation of a th

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