Lab 4/5 results

The Southern blots and plaque lifts were exposed to X-ray film for periods of 5 days and 7 days. The 5 day film gives better results for the Southern blots, while the 7 day film is better for the plaque lift results. The outlines of the membranes are shown on the 5 day film – the 7 day plates can be identified by matching them up to the 5 day film.

Southern blots

2 Southern blots had positive results – the “RT JS” (Group 13) blot at the top left corner of the 5 day film, and the “AC” (Group 8) blot in the top centre. You may use either blot for your results. Make a note of which blot you are using.

Before the sizes of the bands visible on the blot can be worked out, a standard curve must be prepared using the molecular weight markers on the gel that the blot was prepared from. Gel bands migrate differently on each gel, so a standard curve must be prepared for each individual gel. The gel photo for “RT JS” (Group 13) is here, and the gel photo for “AC” (Group 8) is here.

The “RT JS” (Group 13) blot has the wells located at the top of the blot, with the EcoRI digest on the left, and the BamHI digest on the right. The bottom of the blot is the end of the gel.

The “AC” (Group 8) blot has the wells located at the bottom of the blot, with the EcoRI digest on the right, and the BamHI digest on the left. The top of the blot is the end of the gel.

Because the gel photo is not exactly the same size as the blot, the distances travelled by the bands on the blot must be corrected for the differing sizes of the two images. (The blot is the actual size of the gel, while the photograph is smaller than the gel.) If a fluorescent ruler had been included in the gel photograph, this would have provided a “real size” scale for the photo image. Unfortunately, both of the gels which produced good blots were photographed without a ruler. Because of this, a mathematical correction will have to be worked out. For the blot you decide to use, measure the distance from the wells (at one thin end of the blot) to the other end of the blot. Then measure the distance from the wells to the end of the gel on the matching gel photo. (The end of the gel is visible as a change in the shade of grey on the image.) Divide the distance from wells to end on the gel photograph by the distance from end-to-end of the blot. When you multiply the distance from wells to bands on the blot by this correction factor, you will know the location of the positive band on the gel photograph. The corrected distance can then be converted to a size in kilobases using the standard curve prepared using the molecular weight markers.

If you are able to download and manipulate the .jpg images, an alternative way to correct for the different image sizes would be to resize one of the images so that the distance from the well to the end of the gel is the same on both images. The distances measured could then be compared directly.

Plaque lift

The best results for the plaque lift are the “RJ” (Tuesday Group 13) plate, in the bottom centre of the 7 day film. Count the number of positive clones on the film, and then count the number of plaques on a segment of the original plate – stored in the right refrigerator (top shelf, left front). Use these to estimate the percent of positive clones out of the total number of plaques on the plate.

Lab 6 results

For groups that did not get any results for one or both of the PCR reactions, here is a sample photograph of a gel that Mark Sidney prepared. From left to right, the lanes contain 5 µl 1 Kb Ladder, 3655 primer control, 3655 primer PCR, 10 µl 1 Kb Ladder, 3796 primer control, 3796 primer PCR, and 5 µl 1 Kb Ladder.