Expt007 : Double digest of a linear DNA fragment

Method

For this experiment, as in a real laboratory, you need to be really organised and precise especially when labelling the tubes where we are going to keep our samples, or the ones we use for the reactions. You need a sheet of paper and a pen for this experiment, or you can print the sample table provided .

Load Expt007. There are two samples. Sample 1 is the size marker we have used already, and Sample 2 is an 8kb (8000 bp) DNA fragment we are going to analyse.

In this example we are going to work with EcoRI and BamHI.

• Load some DNA from Sample 1 and Sample 2 into reaction tubes

• Analyse Sample 1 and Sample 2 by Agarrose Gel Electrophoresis

• You should see the size markers in track one and an 8kb fragment in track two.

• Close the Agarose Gel Electrophoresis module

• Discard the reaction tubes

• Load some DNA from Sample 2 into a reaction tube.

• Start the Restriction Enzyme Digest module.

• Choose EcoRI .

• Digest.

• Close the Restriction Enzyme Digest module
• Drag some DNA from the reaction tube into the Sample 3 tube to save it .
• Record in your "lab book" that Sample 3 contains DNA from Sample 2 digested with EcoRI, or rename the sample to a more appropriate name.
• Discard your reaction tubes.

• Load some DNA from Sample 2 into a reaction tube.

• Start the Restriction Enzyme Digest module.

• Choose BamHI.

• Digest.

• Close the Restriction Enzyme Digest module
.
• Drag some DNA from the reaction tube into the Sample 4 tube to save it .
• Record in your lab book that Sample 4 contains DNA from Sample 2 digested with BamHI.
• Discard you reaction tubes.

• Load some DNA from Sample 3 into a reaction tube.

  NB: This is DNA we have already digested with EcoRI. We are going to digest it with BamHI as well - this is called a double-digest - i.e. digested with 2 enzymes!

• Start the Restriction Enzyme Digest module

• Choose BamHI

• Digest.

• Close the Restriction Enzyme Digest module
• Drag some DNA from the reaction tube into the Sample 5 tube to save it
• Record in your lab book that Sample 5 contains DNA from Sample 3 digested with BamHI: i.e. it is some of the sample 2 digested with BamHI and EcoRI.
• Discard your reaction tubes

• Load DNA from Samples 1 to 5 into reaction tubes.

• Start the Agarose Gel Electroporesis method

• Load the DNA from the reaction tubes into appropriate tracks: the gel runs instantly

Results and Discussion

Samples 1 to 5 have been loaded into tracks 1 to 5 respectively. Diagram 2 represents the gel obtained.

The table below, shows the observations we have recorded in our notes while we were doing the experiment.

Some fragments present in the track 3 (single digest by EcoRI) are still present in track 5 (double digest EcoRI - BamHI). These fragments do not contain any BamHI sites. (Diagram 3) Diagram 3	Some fragments present in the track 4 (single digest by BamHI) are still present in track 5 (double digest EcoRI - BamHI). These fragments do not contain any EcoRI sites. (Diagram 4) Diagram 4
Some fragments present in the track 3 (single digest by EcoRI) are not present in track 5 (double digest EcoRI - BamHI). These fragments have been cut by the second enzyme: they contain at least one site for BamHI. (Diagram 5) Diagram 5	Some fragments present in the track 4 (single digest by BamHI) are not present in track 5 (double digest EcoRI - BamHI). These fragments have been cut by the second enzyme: they contain at least one site for EcoRI. (Diagram 6) Diagram 6

Your Turn

1) Could you try to draw a simple map of this fragment with the positions of the EcoRI and BamHI sites?

2) The biggest fragment in the BamHI digest contains 2 EcoRI sites. It is indeed cut into 3 smaller fragments while the other one is not cut. Does it contain any other restriction sites?

3) The smallest fragment of the EcoRI digest does not contain any BamHI site. Does it contain any other restriction site?

For the last two questions try all the enzymes provided in the Restriction Enzyme Digest module (restrict yourself to the list of common enzymes!). Trick: cut a gel slice of the desired fragment and save it in a new sample tube before you study it!

You will find the answers here.