Already have an account? Get multiple benefits of using own account!
Login in your account..!
Remember me
Don't have an account? Create your account in less than a minutes,
Forgot password? how can I recover my password now!
Enter right registered email to receive password!
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?)
Explain Requirements and Recommended Dietary Intake? A very small daily intake of vitamin C (10-15 mg/day for an adult) is required to avoid deficiency and stave off scurvy. Ho
Oseltamivir (Tamiflu) This oral neuraminidase inhibitor, started within 36 hours of symptom onset, can decrease the severity and duration of symptoms caused by either influ
Genetic Defect in Pyruvate Dehydrogenase A defed in any of the protein subunits of PDH can result in decrease or complete loss of activity. Severe cases are usually fatal. Sy
What is nervous system
In patients who have not received prior antibiotics and who will ultimately have blood culture positive endocarditis, it is likely that 95 - 100 per cent of all cultures obtained w
How does the sodium-potassium pump present in the cell membrane work? What is the importance of this protein for the cell? The sodium-potassium pump is the transport protein th
Q. Define the Glucose Tolerance Test? In Glucose Tolerance Test (GTT) if the blood sample is to be taken in the morning, patient is advised to follow fasting instructions i.e.
Stability of Natural Colorants in Foods Many attempts have been made to retain colouration by adding chemicals or modifying processing conditions. Ascorbic acid has been claime
Define measuring body composition- Multiple isotope Dilution? Multiple isotope dilution (Total and extracellular body water): It is based on the principle that certain substanc
Explain radiation sterilization Various studies have been conducted which show the effect of processing on vitamins especially, thiamine. In one study, which compared the effec
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!
whatsapp: +1-415-670-9521
Phone: +1-415-670-9521
Email: [email protected]
All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd