User:Brian P. Josey/Notebook/2009/09/30

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DNA Gels

Checked the DNA from yesterday with Anthony. To do this we made a gel. When you add the DNA and the dye (which is one fifth of the volume of the DNA solution) you have to ensure that the pipette tip is not held up too highly, resulting in the dye not landing in the well, and not to deeply, where the gel will be punctured.

Ant's Findings and Questions

Anthony Salvagno: Great job setting the gel up. I imaged it and here are your results:
What does this mean? Well it means the ligation was unsuccessful. The reason I know this is because the two bright bands are supposed to add up to get another band above both. If you look at the ladder (lane 1) the brightest band there is 3kb. The ligation would yield a band halfway between that band and the next band on the ladder (4kb) because the addition of the two bands (one is 1.1kb and the other is 2.5kb) would be 3.6kb. If you also notice there is a very dim band in between our two bands, I think I figured out what that band is. You can look at my notebook for the answer, but I ask you to try and figure it out first. I will give you some hints:

  • I digested pBR322 with EarI
  • I gel extracted the larger piece of DNA from this gel.
    • That entails physically cutting out the slice of DNA from the gel and running that protocol you saw me doing yesterday.
  • Then we ran the ligation.
  • This website could be of use: plasmid Review

Also I think it would be good of you to label that picture I uploaded (of today's gel). This way you can easily describe to someone exactly what they are looking at. You don't need to do anything fancy, but a detail in here of what the lanes are and important bands (for instance the bright band in the ladder is 3kb) would be a good practice. Anyways good job Brian and I'm sorry this didn't work out.

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