Discussion:
Diagnostic Restriction Digests
THE PROBLEM
you have decided to study DNA repair and recombination in E.coli. your best buddy from graduate school sends you a clone of the recA gene. she has told you that the gene is included in 3.266 Kb Bam HI genomic fragment that she has cloned into the pBC vector. however, she has neveer checked the orientation of the insert in the plasmid, and you must determine it using restriction mapping.
FIRST:
obtain the sequence of the E.coli Bam HI genomic fragment that contains the recA gene. the NCBI homepage is a good place to start: https://www.ncbi.nlm.nih.gov/.
SECOND:
let the computer generate the restriction enzyme sites for you! there are many sites with tools that will do this. one is the Saccharomyces Genome Database:https://www.yeastgenome.org/. click on :sequence analysis and tools" and paste the E.coli sequence into the "Yeast Genome Restriction Analysis" to generate a map.
using the results, draw the restriction map of the 3.266 Kb fragment containing the recA gene including the following enzyme sites:
Alu I, Ava II, Bgl II,Cla I,DraI, Hind III, Pvu II, Pst I, and Eco RI
mark each site with its appropriate location relative to the 5' Bam HI. for example, assign the 5' Bam HI site as position 1, and so on. draw the coordinates of the recA gene onto the restriction map.
Next, draw out the two different plasmid maps that would result from inserting this 3.266 Kb Bam HI fragment into the pBC vector in opposite orientations. include the following enzyme sites on your plasmid maps:
Determine the sizes of the restriction fragments that you would expect ot obtain from the following digests:
Bam HI
Eco RI
Bam HI and Eco RI double digest
Cla I
List the sizes for both plasmid orientations. you do not consider cuts using any of the enzymes besides those listed above, but of course they need to be on your original map so that you can predict the fragment sizes.
EXTRA CREDIT:
Determine the Ava II digest results for both plasmids.
ANSWER THE FOLLOWING QUESTIONS;
1. which enzyme is better to use for determining the orientation of the insert, Cla I or Eco RI?
2. could Alu I be used for determining the orientation of this insert?
3. could Hind III be used for determining the orientation of this insert?
LASTLY,
go to the new england biolabs web (www.neb.com) since you have limited funds for research, you are concerned with reagent costs. which restriction enzymes would provide you with the most cost-effective solution to your problem. Ava II, Bam HI, Eco RI, or Cla I?
Note: one unit of restrictin enzyme is defined as the amount of enzyme needed to completely degest 1 microgram DNA in 50 Microliter volume in one hour.
Solution Summary
quences