1. Prepare a 40 ml 1 % agarose gel in 0.5X TBE, containing 0.5 µg/ml ethidium bromide. Each gel will be shared by 2 groups on the same bench.
2. Transfer 15 µl aliquots of your PCR and control reactions to fresh 1.5 ml tubes. Add 1.5 µl of 10X loading buffer to each sample. Mix by flicking tube and pulse spin on microcentrifuge to collect sample in bottom of tube.
3. Each group will load PCR and control samples on one side of a central molecular weight markers lane containing 5 µl of 1 Kb ladder.
4. Run gel for 45-60 minutes at 100 volts, or until bromothymol blue band is about 2/3 of the way down the gel. Photograph the gel, and dispose of it in the ethidium bromide waste.
|
Step |
Time |
Temperature |
Activity |
|
1 |
2 minutes |
94°C |
denaturation |
|
2 |
30 seconds |
94°C |
denaturation |
|
3 |
30 seconds |
55°C |
annealing |
|
4 |
1 minute |
72°C |
extension |
|
2-4 |
|
|
repeat for 30 loops |
|
5 |
5 minutes |
72°C |
extension |
|
6 |
indefinite |
4°C |
hold |
1. Prepare a standard curve for the molecular weight markers from the photograph of your PCR product gel. (Label the graph properly with a title, units, and explanation of any symbols used.) Be sure to identify the proper molecular weights of the 1 Kb ladder bands on your gel.
2. Use the standard curve to work out the size of the band(s) present in the PCR and control lanes of both group’s samples. Results should be reported to the proper number of significant figures for the experimental results (i.e., the number of significant figures in your measurement of the distance of the band(s) from the well.) Show sample calculations. Compare the size of the bands calculated from the standard curve with the expected size of the PCR products for each group. (These can be calculated by subtracting the difference between the numbers for the primers used.)
3. Why was a negative control done for each PCR reaction? What could cause band(s) to show up on the gel lane loaded with the negative control?
Each group that provided Southern blots and/or plaque lifts for probing in the last lab should identify the X-ray films prepared from their blots and/or plaque lifts and label them so they can be aligned with the gel photographs and/or stored phage plates. (Gel photos and phage plates will have to be shared among groups, so do not dispose of them.)
1. Choose one of the sample X-ray films prepared from a Southern blot that contains visible bands. Record which group’s results you are using. Use the ruler on the gel photo to prepare a standard curve from the 1 Kb Ladder molecular weight markers. (Label the graph properly with a title, units, and explanation of any symbols used.) Be sure to identify the proper molecular weights of the 1 Kb ladder bands on your gel.
2. Measure the distance from the wells to the visible bands on the X-ray film. Use the standard curve to calculate the size of the bands that hybridized to the probe. Results should be reported to the proper number of significant figures for the experimental results (i.e., the number of significant figures in your measurement of the distance of the band(s) from the well.) Show sample calculations.
3. Choose one of the sample X-ray films prepared from a plaque lift. Record which group’s results you are using. Count the number of positive clones present on the plate. Align the X-ray film with the stored plate to identify the position of the positive clones on the plate. Estimate the number of plaques on the plate, and calculate what percentage of the plaques contain sequences homologous to the probe used.
4. If you want to clone a specific gene from a new organism, why would you want to probe a Southern blot before you make and screen a genomic library?
5. What was the probe used to screen the blots and plaque lifts? Describe the function of the steps involved in labelling the probe.