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

draw the cell at metaphase, Consider cell with a diploid chromosome number...

Consider cell with a diploid chromosome number of 4 (2n=4), one pair of the chromosomes is metacentric,the other is acrocentric. The metacentric is labeled a 1 a 1 while the acr

Major conventional symbols & signs used in genetic family, What are the maj...

What are the major conventional symbols and signs used in genetic family trees? In the genetic family trees the male sex is usually represented by a square and the female by a

WHY ARE WALLS OF THE LEFT VENTRICLE THICKER THAN THOSE RIGHT, Ask question ...

Ask question #Minimum 250 words accepted#

Respiration in fishes, RESPIR A TIO N IN FISHES - Respiratory org...

RESPIR A TIO N IN FISHES - Respiratory organs are gills. In elasmobranchi 5-7 pairs of gills present, spiracles present. In Teleostomai 4 pairs gills present, covered

Why the ph of urine can be quite variable, The ph of urine can be quite var...

The ph of urine can be quite variable. Why? Why is bromothymol blue used as a ph indicator instead of phenal red when titrating blood?

Relate osseointegration with biointegration, Osseointegration Vs. Biointegr...

Osseointegration Vs. Biointegration In 1985, dePutter observed that there are two ways of implant anchorage or retention: mechanical and bioactive. Mechanical retention refers

Regulation of egg, difference between mosaic and regulation egg

difference between mosaic and regulation egg

Which is plant tissues specialized in covering, Which is plant tissues spec...

Which is plant tissues specialized in covering? The dermal tissues or covering tissues of the plants are the epidermis (that covers the leaves and the young stems and shoots) a

Cockroach heart, How last chamber of heart in cockroach receives blood with...

How last chamber of heart in cockroach receives blood without having ostia?

Mountain forest ecosystem as life support systems, Mountains and their fore...

Mountains and their forest ecosystems are regarded as the water towers of the world. A good example is the extraordinarily massive Himalayan Mountains which have shaped the climate

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