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

Write the meaning of polyuria, Q. Write the meaning of Polyuria? Increa...

Q. Write the meaning of Polyuria? Increased Urine (Polyuria): When sugar is increased in the blood it is filtered from the body along with the water. This is the reason for fre

Why is drosophila a convenient animal study of linked genes, Why is drosoph...

Why is drosophila a convenient animal for the study of linked genes? The fruit fly drosophila is appropriate for the study of Genetics because it presents many distinct traits

Explain the mitotic prophase, Which of the following best explains what wou...

Which of the following best explains what would happen if recombination (crossing-over) happens during mitotic prophase? A. Recombination during mitosis will lead to segregatio

Which bound to hemoglobin, Which of the following are bound to hemoglobin w...

Which of the following are bound to hemoglobin when hemoglobin is in the R-state? Choose all that apply. 1. Fe2+ 2. CO2 3. 2,3-bisphosphoglycerate 4. Fe3+ 5. Oxygen

Genic type-genetic basis of sex determination, Genic Type-Genetic basis of ...

Genic Type-Genetic basis of Sex Determination In some organisms, certain indepe~ident genes located on different chromosomes are responsible for sex determination. Most species

Which disease is associated with rapid dehydration, Which disease is associ...

Which disease is associated with the following symptoms? Sudden onset of profuse watery stool followed by vomiting, rapid dehydration and muscular cramps?

Explain ketogenic diet, Explan Ketogenic diet Ketogenic diet:   It is ...

Explan Ketogenic diet Ketogenic diet:   It is occasionally used to facilitate the control of epilepsy. Here, the patient  is  initially  fasted  for  48 hours and thereafter,

Define isolated soybean proteins in dairy type products, Define isolated so...

Define isolated soybean proteins in Dairy type products? Soybean protein isolates are used in non-dairy coffee whiteners, liquid whipped toppings, emulsified sour cream or chee

Metabolism of fructose, Fructose is an abundant sugar in the human diet; su...

Fructose is an abundant sugar in the human diet; sucrose (table sugar) is a disaccharide that when hydrolyzed yields glucose and fructose and fructose is also a major sugar in hone

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