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Virtual-Lab Exercise Southern Blot Analysis Figure 1: Shown above is an image displaying the sizes of the unique bands present in the 1kB ladder that is used during this weeks lab

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Virtual-Lab Exercise Southern Blot Analysis

Figure 1: Shown above is an image displaying the sizes of the unique bands present in the 1kB ladder that is used during this weeks lab. The ladder was loaded on an agarose gel containing ethidium bromide and separated by agarose gel electrophoresis. Visualization was by exposure to UV light and imaging.

Figure 2: Genomic DNA from individual plants was PCR amplified using specific primers designed to amplify the presence of an insertion. Lanes are as follows: Ladder, 1kb ladder (Genedirex); -ve, no template DNA; +ve, PCR amplified positive control using the pROK2 plasmid as the template; A to C, PCR amplified products using DNA extracted from individual plants.

Question One:

Describe why we need both a negative and a positive control for the PCR reaction. What can you interpret from the results for each sample?

Question Two:

Confirm the size of your insert. In order to do this, you will need to graph the distance travelled from the well for each fragment on the x-axis vs the size of each fragment on the y axis. As mentioned in class if we plot the distance each marker travels vs the known size of each fragment on a semi-log graph we should end up with a linear standard curve using a semi-log graph.

The easiest way to this is in excel. Instructions for how to do this are readily available on the web. We’ve linked one here How to Create a Semi-Log Graph in Excel (statology.org).

You should use “mm” and not “cm” as your unit of measure for distance to make it easier to read the graph. You can change the number of lines displayed in the graph by right clicking the numbers on each axis. The more units you display the easier it will be to tell the size of the fragment.

What is the size of the fragment?

Based on the graph is your PCR amplified fragment the expected size? Is the graph linear? If not, why might that be the case?

Question Three:

You are asked to measure the OD 260/280 of your amplified product to determine the concentration.

Why do we look at the OD 260/280 ratio? What does it tell us?

What would be the significance of the following OD 260/280 ratios in the context of this lab? 1.8

2.0

1.6

Question Four:

You have a stock DNA sample of 1,950ug/mL that must be diluted to a final concentration of 20ng/uL in a 20uL reaction for a restriction digest. See the reaction below. How would you do this?

10X Buffer 2 uL Water ? uL

DNA ? uL

EcoRI 1(U/ul) 1 uL Total 20uL

Virtual-Lab Exercise Southern Blot Analysis Figure 1: Shown above is an image displaying the sizes of the unique bands present in the 1kB ladder that is used during this weeks lab

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Virtual-Lab Exercise Southern Blot Analysis Figure 1: Shown above is an image displaying the sizes of the unique bands present in the 1kB ladder that is used during this weeks lab

Biology

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