Alice:LabNotes/2007-11-26
Run PCR purification with post-PCR product from the 6 failed sample from DNA sequencing, which was the original sample from well #1,2,3,12,14,16. The method used this time is different from the method using column. This method is more old school way that takes
longer but allows greater %yield. It is good if one wants to preserve greater amount of DNA.
Purify the post-PCR samples with EXOsap-IT, and then run a gel to check if all the reaction worked fine.
Each well contain total of 5 ul of mixture of reagents.
Summary of volumes of Reagents used:
' | well 1 | well 2 | well 3 | well 4 | well 5 | well 6 | well 7 | well 8 | |
Low Mass bp Ladder | 3 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | |
100 bp Ladder | 0 ul | 3 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | |
TBE buffer | 1 ul | 1 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | 0 ul | |
Novex 5x TBE loading dye | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | |
post-PCR sample | 0 ul | 0 ul | 4 ul | 4 ul | 4 ul | 4 ul | 4 ul | 4 ul | |
Results:
File:ZhangLab 2 2007-11-26 13hr 17min-2-.png
This could have been a rather good gel if the focus of the gel imaging machine could be adjusted to be either further away of closer to the level of samples. The gel was stained with SYBGold for 15 min. Even though the gel is fuzzy, but it is easy to tell that the intensity seems to be desirable, since all the bands are visible. Well #3, #4, and #6 shows weaker intensity maybe because the reaction didn't shoot up until very late stage, so they are not as desirable as the other three. I also ran a gel with the same setup with different stain SYBE safe, but the result didn't turn out to be much more different. However, it is very likely that I deleted the image by accident, otherwise could have put it up for comparison.
I also ran another two gel with post-PCR product (rs1264899) of sample 14, just to make a comparison between agrose and PAIGE gel. The image is shown below:
File:ZhangLab 2 2007-11-26 14hr 29min.png
File:ZhangLab 2 2007-11-26 14hr 36min.png
The top image is from agrose gel, and the bottom image is from PAIGE gel.
Summary of volumes of Reagents used:
' | well 1 | well 2 | well 3-6 | ||
Low Mass bp Ladder | 3 ul | 0 ul | 0 ul | ||
100 bp Ladder | 0 ul | 3 ul | 0 ul | ||
TBE buffer | 1 ul | 1 ul | 1 ul | ||
Novex 5x TBE loading dye | 1 ul | 1 ul | 1 ul | ||
post-PCR sample | 0 ul | 0 ul | 3 ul | 3 ul | |
Results Clearly, these gel had similar results, but the focus of the machine was very bad. Therefore, we can conclude that so far there should be nothing wrong with the stain, sample, or the gel. Improving my skill to adjust the machine should be able to solve the problem.