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

Is this refers to the mn and abo loci mentioned in class, This problem refe...

This problem refers to the MN and ABO loci mentioned in class. It also refers to the Rh locus, which is responsible for the positive/negative part of the blood type. The Rh+ allele

Explain deet, DEET DEET is available in the US in many formulations wi...

DEET DEET is available in the US in many formulations with concentrations of 5%-40% and 100%; higher concentrations present complete protection for a longer period of time, bu

Etiological factor of diverticular disease, Q. Etiological factor of divert...

Q. Etiological factor of diverticular disease? The causes of diverticular disease are not certain, but several factors may contribute to changes in the wall of the colon. These

Meiosis, does crossing over occur in all the homologous chromosomes???

does crossing over occur in all the homologous chromosomes???

Describe ecological phytosociological, Q. Describe Ecological phytosociolog...

Q. Describe Ecological phytosociological? Field studies are desirable for an understanding of the relationship of any group of plants. If it is not possible to study the plants

Nervous tissue, This constitutes about 2.4% of the body weight of man viz. ...

This constitutes about 2.4% of the body weight of man viz. Brain-1400 gram Spinal nerve-150 gram Spinal cord-30 gram Cranial nerve-10 gram C.S.F. is altogether not present in

Male reproductive system - urethera, URETHRA - It is common duct as ...

URETHRA - It is common duct as sperms & urine both pass from it. It receives juices of prostatic gland & cowper's gland. Urethra is 20 cm long, passes through the peni

Explain the characteristics of starch granule, Explain the Characteristics ...

Explain the Characteristics of Starch Granule Starch granules, primarily, are made up of amylose (20-30%) and or amylopectin (70-80%) molecules arranged radially. Each granule

Explain the activity of decomposers, Why are producers the first trophic le...

Why are producers the first trophic level to advantage from the activity of decomposers? Decomposers return nutrients in dead tissues and wastes to the soil or water; producers

For non-insulin users consumption of alcohol, Q. For non-insulin users cons...

Q. For non-insulin users consumption of Alcohol? 1. Substitute for fat calories. A drink providing 100 calories should be substituted by 2 fat exchanges. . 2. Avoid if your

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