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

Deficiency diseases-thiamine deficiency (hypothiaminosis), Thiamine deficie...

Thiamine deficiency (hypothiaminosis) Thiamine deficiency is characterized by signs of nervous system. The primary  thiamine deficiency is not common in animals. However, seco

Describe organs of female reproductive system, Q. What are the organs that ...

Q. What are the organs that are part of the female reproductive system? The organs that constitute the female reproductive system are the ovaries, the uterus, the Fallopian tub

What is the process of protein synthesis, Q. After the fact that it is base...

Q. After the fact that it is based on information from mRNA what is the process of protein synthesis called? Protein synthesis is called translation of genetic information into

Are there differences between the male and female skeletons, Q. Are there d...

Q. Are there differences between the male and female skeletons? Many general differences exist between female and male skeletons. Male skeleton is normally larger and heavier t

Explain the future challenges for neuroscience, Explain the Future challeng...

Explain the Future challenges for Neuroscience? When the analysis of single cells and even networks has progressed very nicely, there still remain many significant questions. A

Explain somatic cell, For a somatic cell with 2n = 4, which of the followin...

For a somatic cell with 2n = 4, which of the following is true? (Note: G1- growth phase 1, G2 - growth phase 2, M - metaphase, P - prophase and T - telophase) a.  (Number of

Glycogenesis, Glycogenesis :   This  is an anabolic process  in which  gluc...

Glycogenesis :   This  is an anabolic process  in which  glucose is polymerized  into glycogen by the   sequence of  reactions.  Since glycogenesis occur  in all  body  cells .but

Explain phylogenetic or cladistic classification, Phylogenetic or Cladistic...

Phylogenetic or Cladistic Classification Phylogeny plays a great role in classification. It is the  appropriate theoretical background for taxonomy and is quite essential in exp

Ozone layer play in the evolution of early life on earth, What role did the...

What role did the appearance of the ozone layer play in the evolution of early life on Earth? Ultraviolet radiation from the sun damages DNA, but the development of the ozone l

What is the curvature of the lens, What is the curvature of the lens I...

What is the curvature of the lens In a human being and most other mammals, the first three mechanisms are not possible and accommodation occurs by the fourth mechanism only. T

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