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

What an enzyme is called, The substance that is worked on by an enzyme is c...

The substance that is worked on by an enzyme is called: a. product b. coenzyme c. substrate d. inhibitor

Difference between lamarckism and neo-lamarckism, DIFFERENC E BETWEEN LAMA...

DIFFERENC E BETWEEN LAMARCKISM AND NEO-LAMARCKISM - S.No Lamarckism Neo-Lamarckism 1. It refers of the original

Regernation, Give ditail account on regernation in invertebrates and verta...

Give ditail account on regernation in invertebrates and vertabrites

Enumerate about the luria nebraska battery, Enumerate about the Luria Nebra...

Enumerate about the Luria Nebraska battery Luria Nebraska battery will be based on the assumption that the only connecting link between Luria and that procedure is the set of

What are the role of mr, MR means medical representative. Medical rep has t...

MR means medical representative. Medical rep has to sell products of pharma companies or drug manufacturers. He has to go to doctors and medical shops to sell drugs and promote dru

What is growth monitoring of an infant, What is Growth Monitoring? In t...

What is Growth Monitoring? In third world countries, about half the children are short and underweight for their age. Inadequate nutrient intake is the main reason. Inadequate

How common are coiled-coil motifs to each other, 1. Name four experimental ...

1. Name four experimental techniques to determine protein-protein interactions other than X-ray & NMR techniques. 2. How common are Coiled-coil motifs? Where are they found? Giv

Glycogen degradation and glycogen synthesis , If glycogen degradation  and...

If glycogen degradation  and glycogen synthesis were allowed to occur concurrently,  the  net  effect  would  be  hydrolysis  of  UTP,  a  so-called  futile  cycle. To stop this, b

Mechanoreceptors - receptors, Mechanoreceptors - Receptors Mechanorece...

Mechanoreceptors - Receptors Mechanoreceptors involve those receptors involved in perception of touch, pressure, tension, hearing, vibration, gravity, muscle tension etc. Th

What is exocytosis, Which of the following is true for exocytosis? A. D...

Which of the following is true for exocytosis? A. During exocytosis in skeletal muscle cells, there will be release of calcium ions from intracellular vesicles in the sarcoplas

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