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 casparian strip, Casparian strip  In plants, the impermeable waxy ...

Casparian strip  In plants, the impermeable waxy layer between the cells of endodermis which stops water and solutes from entering into the xylem, except by passing through the cyt

Tasks of orientation phase - phases of nurse, Tasks of  Orientation Phase: ...

Tasks of  Orientation Phase: Establishing contact with the patient  Developing  the pactlcontract  Talking to the patient  Establishing Contact with the Patient

Pollen stigma interaction - stigma, Pollen Stigma Interaction - Stigma ...

Pollen Stigma Interaction - Stigma The Stigma After landing on the stigma pollen grain germinates, and produces a pollen tube that carries the male gametes. The stigma has bee

What electrocardiogram results show a shortened qt interval, Electrocardiog...

Electrocardiogram results show a shortened QT interval and an increase in the P-R interval (i.e., decreased duration of action potential and decreased conduction velocity). Laborat

Pollenkitt - pollen wall, Pollenkitt - Pollen Wall The lipoidal layer ...

Pollenkitt - Pollen Wall The lipoidal layer found on the outside of the mature pollen grains of many insect pollinated species is known as the pollenkitt. In addition to lipid

What do you know about tricuspid stenosis, Q. What do you know about Tricus...

Q. What do you know about Tricuspid stenosis? It is rare disease, with rheumatic etiology seen in 90 per cent of cases. In patients with rheumatic mitral stenosis only 3-5 per

What happens to the parent cell, What is produced at the end of the cell cy...

What is produced at the end of the cell cycle? how do they compare to each other and to the parent cell? What happens to the parent cell?

Define observation or inference for mucic acid test, Define Observation or ...

Define Observation or Inference for Mucic Acid Test? 1. White crystals of mucic acid will be seen. 2. The sugar solution contains galactose. 3. Lactose gives this test, s

Define criteria for assessment of riboflavin status, Define Criteria for As...

Define Criteria for Assessment of Riboflavin Status? Riboflavin status can be assessed by measuring urinary excretion of the vitamin in fasting, random, and 24-hour specimens o

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