Talk:20.109(F09): Mod 1 Day 3 Agarose gel electrophoresis
Here is an example of a gel with all the expected bands, as well as a gel with the needed bands excised.
You can use these in your lab report if you so choose.
T/R lab
| Red | Orange | Yellow | Green |
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| Blue | Purple | Pink | |
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And here's the gel with your recovered fragments/backbones, ready for ligation.
| Lane | Sample | Lane | Sample |
|---|---|---|---|
| 1 | Blue Frag | 6 | Pink BKB |
| 2 | Blue BKB | 7 | -- |
| 3 | Purple Frag | 8 | -- |
| 4 | Purple BKB | 9 | -- |
| 5 | Pink Frag | 10 | ladder |
| Lane | Sample | Lane | Sample |
|---|---|---|---|
| 1 | Red Frag | 6 | Yellow BKB |
| 2 | Red BKB | 7 | Green Frag |
| 3 | Orange Frag | 8 | Green BKB |
| 4 | Orange BKB | 9 | -- |
| 5 | Yellow Frag | 10 | ladder |
Red Team: Yours didn't show up well. Yours was the first sample I loaded and the gel was cracked so the sample went through the bottom of the gel. I tried to pipette as much of the sample as possible out of the broken gel and into this gel, but I didn't get much. Assume that your sample is about like that of Orange to do your HW due on Thursday.
Red Team: I re-ran 5 ul of your product with 10 ul water and 2 ul loading dye. I ran it for 20 min at 125V.
Please use this image for your final lab report.
| Lane | Sample |
|---|---|
| 1 | 10 ul ladder |
| 2 | 5 ul backbone + 10 ul H20 + 2 ul loading dye |
| 3 | -- |
| 4 | 5 ul fragment + 10 ul H20 + 2 ul loading dye |
W/F Lab
| Red | Green | Blue |
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| Purple | Pink | Pink Post-stained fragment part |
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Green Group: For some reason, your ladder did not show up properly. Please use the example images at the top of the page for your calculations for next time.
Pink Group: Unfortunately, it appears that you didn't have a PCR product. We had discussed possibilities of why we didn't see a band at first, namely the EtBr migrating out of the gel. This was a good hypothesis since we couldn't see the last few bands of the ladder. While you were recovering the backbone, I post-stained the gel in an EtBr / TAE solution for 30 min followed by two rinses in DI water. I cut through the ladder, but we can now see the 500 bp band, but still can't see a fragment. Since we can see the 500 bp band, it means that your band was not run off of the gel. You will use fragment from the Purple group, so use that image for your calculations for next time.
And here's the gel with your recovered fragments/backbones, ready for ligation.
| Lane | Sample | Lane | Sample |
|---|---|---|---|
| 1 | ladder | 8 | Blue BKB |
| 2 | Red BKB | 9 | Blue Frag |
| 3 | Red Frag | 10 | Pink BKB |
| 4 | Green BKB | 11 | Purple BKB |
| 5 | Green Frag | 12 | Purple Frag |
| 6 | -- | 13 | -- |
| 7 | ladder | 14 | ladder |
