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

Pumps, The pumps The blood from the operating field is sucked with t...

The pumps The blood from the operating field is sucked with the help of the cardiotomy sucker and pump, which is filtered to remove all particulate matter and then pass

Father, who is the father of science

who is the father of science

Corelation of st depressoin with coronary angiography, Q. Corelation of ST ...

Q. Corelation of ST Depressoin with Coronary Angiography? Various investigators considered 1 mm of horizontal or downsloping ST depression to denote a positive test and used fr

Can you explain the anoxia, Q. What is the anoxia? Anoxia is a situatio...

Q. What is the anoxia? Anoxia is a situation in which there is no available oxygen in the cell without oxygen the respiratory chain stops there is no ATP production the cell do

Preserving foods, One of man's oldest methods of preserving foods is drying...

One of man's oldest methods of preserving foods is drying or desiccation. The preservation of foods by drying is a direct result of removal of moisture. Microorganisms need water

Asymptomatic or mildly symptomatic patients, Q. Asymptomatic or Mildly Symp...

Q. Asymptomatic or Mildly Symptomatic Patients? The treatment is controversial. Some give them betablockers/or calcium channel blockers like verapamil, in the hope of preventin

Explain the hydrophobic grid membrane filter (hgmf), Explain the Hydrophobi...

Explain the Hydrophobic Grid Membrane Filter (HGMF)? It is also used to enumerate microorganisms from a variety of food samples. By using this method, organisms as few as 10 ce

Define solubility affecting absorption of dietary iron, Define Solubility a...

Define Solubility affecting absorption of dietary iron? Solubility is crucial for non-haem iron absorption as the inorganic iron salts have to be solubilized in the intestine f

Parasite and host during incubation period, Parasite and host during incuba...

Parasite and host during incubation period Incubation period is the second stage in the process of a disease. It is the time between the day of infection to the appearanc

Name the enzyme present in saliva, Name the enzyme present in saliva and sa...

Name the enzyme present in saliva and say what type of food it acts on.   The enzyme in saliva is salivary amylase and it acts on starch.

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