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Examining DNA plasmid restriction digest

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Hi again. I am attaching a document that provides details on a lab to be done dealing with DNA sequencing.
I am looking for assistance in two areas of this lab:
1) p. 67 - questions 1, 2 and 3

These questions ask:
1. how do you predict the DNA bands produced after digesting a recombinant plasmid. How do you know what size your recombinant plasmid is after cloning and ligation of the insert into the plasmid.
2. How do you prepare a master mix to use for a restriction digest.
3. what is a plasmid copy number and how do you estimate the copy number for a plasmid

2) p. 80 (i.e. last page of the lab) -- some ideas,suggestions, notes, reference material for a 5 - 10 minute presentation on the topic in point 8 - Groups 5 and 13: Specificity of the DNA sequencer, Applied Biosystems model 3730.

Let me know if you can help me out within the required timeframe.
Also, please let me know if the credit value is acceptable.

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Solution Preview

Here is the information to help you with both parts of this assignment. The word document contains the information to walk you through answering questions 1-3 along with information about the Applied Biosystems 3730 Sequencer. At the end of the word document you will also find the DNA sequences for your plasmid, insert, and plasmid + insert that you need to answer question 1. In the text I walk you through where these sequences came from so ...

Solution Summary

This explanation will provide you with an understanding of how to determine what size DNA fragments will be produced during a restriction digest. It will explain how you generate a DNA sequence when cloning a specific piece of DNA into a DNA plasmid and how you use the NEBcutter website as well as the NCBI blast website. This explanation will also discuss how to set up a restriction enzyme reaction and describe the concept of copy number and how copy number affects which DNA plasmid you use to do your DNA cloning. This explanation also includes information on DNA sequencing, what it is and how it is accomplished.

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ampicillin-resistant plasmid

You have a 4kb ampicillin-resistant plasmid with one BamHI site and a single HindIII site 1.5kb away from the BamHI site (shortest distance).

you cut one microgram of the circular DNA (which we will assume has no nicks in it) with BamHI, and one microgram with HindIII. You heat-inactivate the enzymes (if you prefer you could purify the DNAs by phenol extraction and ethanol precipitation), mix the two linear DNAs together, denature (by boiling for 2 minutes) and then cool slowly to allow hybridization (these conditions should allow only hybrids with perfect or near-perfect base-pairing). What products would you expect tofind?

If you took a small amount of the product and transformed competent cells, which, if any, of the DNA species would you expect to give rise to transformed colonies? Explain.

Imagine that the plasmid described above has a single EcoRI site (say 1.0kb from the BamHI site and 0.5kb from the HindIII site) and that you had an identical plasmid except for a 1bp change (small enough that it will not affect hybridization properties) that eliminates the EcoRI site (GAGTTC instead of GAATTC).

Design an experiment to test what happens (i.e. what is the final plasmid DNA content of cells in a transformed colony) if a competent cell is transformed with circular double-stranded plasmid DNA where there is a mis-match (e.g. a G-T base-pair instead of an A-T base-pair). Be sure to explain what you do, what you measure and how you interpret the results.

What result do you expect? Explain why you expect that result?

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