Pre-lab assignment 3

 

Name:                                                                                                                                                     Score:                     /5

 

1.  On the server you will find the text file (pET21b-CBL.txt) of our plasmid DNA with the CBL sequence inserted (lower case letters).  This is the same file we used to design our mutants.  Create a new text file that reflects the new sequence of your mutated DNA.  Remember, the DNA we ordered is the reverse complement to the sequence shown.  Save this file using a new name in the drop box folder on the server. 

 

2.  Go New England Biolabs Homepage (www.neb.com)

 

Click on Try Nebcutter

Paste the entire DNA sequence from the pET21b-CBL file

Select "circular" sequence type of analysis 

Click submit.  (the display now shows the circular plasmid and all the restriction sites)

 

a) Make a “custom digest” using the restriction enzyme you ordered, click on view gel under main options, what are the sizes of the three longest fragments?

 

 

b) Make a second  “custom digest” using a restriction enzyme form the list below, click on view gel under main options, what are the sizes of the three longest fragments?

 

 

SmaI, EarI, PstI, EcoRI, KpnI, BstUI,

HaeIII, PhoI, BstNI, BfaI, SfoI, SacII

 

3.  Repeat parts 2a and 2b using your mutant sequence of DNA.  Report the three longest fragments for each digest.

 

 

 

 

Notebook assignment (5 points)

 

Using the "Methods" manual as a guide, design an experimental procedure in which you digest both the parent (unmutated plasmid) and the mutant plasmid with two different enzymes. Consider your reaction conditions, amounts of enzymes and DNA, control reactions you need to run and the agarose gel conditions to resolve the fragments.  You need not design a DNA isolation procedure, we will be using a kit.

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