Assignment for Restriction Enzymes
Sometimes it is necessary to screen a large number of DNA samples to identify one that has a restriction pattern typical of a desired recombinant construction. If you had 100 different DNA samples to screen and you had to add DNA, buffer, water, and enzyme separately, you would need to do at least 400 pipettings. Such a large number would not only use a large number of tips but will also be prone to errors. One way to minimize the number of pipetting actions is to create a master mix of components so that you only need to do a minimum number of pipetting manipulations per sample. When designing master mixes, it is wise to add an extra reactions worth of volume for each 10 reactions to ensure that repetitive pipetting errors do not leave you short of master mix for the last tube.
Below is a table showing the restriction digest components and the volumes needed for each reaction. Select the components needed for the master mix and enter their volume contribution in the Master Mix column.
| Component | Stock | Volume/1 rxn | Master Mix |
|---|---|---|---|
| DNA | 0.25 µg/µL |
8 µL |
|
| 10X Buffer | 10X |
2 µL |
|
| nanopure water | pure |
9.8 µL |
|
| EcoRI | 20U/µL |
0.2 µL |
|
| Total Volumes | 20 µL |
Which component would you add last to the Master Mix?
To which component(s) will the master mix be added in order to complete the set up of the 100 reactions?
Volume of Master Mix needed per reaction =____________________µL.
Such experiments will include a known DNA sample cut with the same enzyme (positive control) and also a sample that contains no enzyme (negative control). If none of the 100 DNAs being screened seem to be digested, but the positive control is cut, could it be because of glycerol inhibition? Explain.
What other explaination could you offer for the negative results?
Below is a picture of DNA fragments from two samples above, separated on an agarose gel.
The positive control is the vector that has been cut with the same enzyme as the samples. The uncut sample can be used to determine if the sample is completely cut or only partially cut. The numbers next to the bands in the standard are in Kb.
Estimate the size of each band in sample1 and sample 2 in Kb.
Sample 1: a_____Kb, b_____Kb, c_____Kb
Sample 2: e_____Kb, f_____Kb, g_____Kb, h_____Kb, i_____Kb, j_____Kb
Which bands in sample 2 represent uncut DNA? ______________________
Band c in sample 1 is not observed in sample 2. Does this mean that the sample 2 is missing one of the restriction enzyme sites or is there possibly another explaination (if so, explain)?