Cloning protocol, Biology

Assignment Help:

1945_Alkaline Phosphate treatment.png

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.

(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?)


Related Discussions:- Cloning protocol

Pyruvate dehydrogenase complex, Pyruvate Dehydrogenase Complex The Pyru...

Pyruvate Dehydrogenase Complex The Pyruvate Dehydrogenase Complex (PDC) is one of the single central enzymes of aerobic metabolism. After completion of this animation you shoul

Explain the functionality of carboxymethyl cellulose, Functionality of CMC ...

Functionality of CMC CMC is soluble in cold water and mainly used for controlling viscosity without gelling. As its viscosity drops during heating, it may be used to improve t

Changes in the malarial parasite, The parasite completes a part of its life...

The parasite completes a part of its life histroy in man in two stages. They are : a) Pre-Erythrocytic cycle. b) Erythrocytic cycle. 1. When an infected female Anopheles mosquit

Explain neurochemical process, Alertness and sleep are dependent on the act...

Alertness and sleep are dependent on the activity of the brain as a whole, although different levels of consciousness are determined primarily by areas of the brain stem. A key ana

Pulmonary ventilation, Pulmonary Ventilation: Pulmonary ventilation is th...

Pulmonary Ventilation: Pulmonary ventilation is the process by which gasses flow between the atmosphere and lung alveoli. Air moves into the  lungs when air pressure  inside the

Glomerular filtrate, Normal 0 false false false EN-IN ...

Normal 0 false false false EN-IN X-NONE X-NONE MicrosoftInternetExplorer4

Cytological approach, Write an essay on cytological approach in taxonomy.

Write an essay on cytological approach in taxonomy.

Why c4 plants are more efficient in photosynthesis, C4 plants are more effi...

C4 plants are more efficient in photosynthesis than C3 plants due to: 1. Higher leaf area 2. Presence of larger number of chloroplasts in the leaf cells 3. Presence of thi

Define role of vitamin d in controlling gene expression, Define role of Vit...

Define role of Vitamin D in controlling gene expression? Vitamin D is a secosteroid that is metabolically activated and degraded through the actions of three cytochrome P450 hy

Define the carbohydrate and fat required for elderly, Define the Carbohydra...

Define the Carbohydrate and Fat required for elderly? Carbohydrate: It is usually recommended that approximately 55en% (percent of  energy) be provided from carbohydrate food

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