User:Anthony Salvagno/Notebook/Research/2010/01/07/Sap14 Gel, SapCap Ligation, and NotI digestion

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Gel Results of Clone 14 Sap Digestion


I finally figured out what those artifacts are, and it is from light getting through my hood hole. If the camera isn't in the proper position then light gets through. I will make some kind of cloth cover with a hole for adjusting.

Anyways this gel puzzled me. Lanes marked as follows:

  1. 1kb ladder
  2. 1kb ladder 2x
  3. 1kb ladder 3x
  4. undigested clone 14
  5. SapI digested clone 14

It looks like SapI didn't do a complete digestion, but then why does the supercoiled piece not run as far as the supercoiled piece in lane 4? Then there is also no nicked DNA in lane 5. If the digestion did cut properly then why is the band not at the same length as the linear band in lane 4? I am going to go with digested DNA and SapCap ligate for now. Koch and I discussed and here is what we decided:

  1. Cut with NotI
  2. Ligate SapCap to Sap14
  3. Run gel with Cap14 (new name), Not14, Sap14, and Clone 14.
  4. Hope for better results.

Ligation of SapCap to Sap14

{{#widget:Google Spreadsheet |key=tFNjYk0_2C77A5_gO_WCswQ |width=700 |height=300 }} Run for 1 hour.

  • After cleaning, the concentration is 14.7nM

NotI digestion of Clone 14

{{#widget:Google Spreadsheet |key=tL9kTdit2w6NLjuCWMygOPw |width=500 |height=300 }}

Gel of it All

Lanes:

  1. 1kb Ladder
  2. Clone 14
  3. Sap14
  4. Cap14
  5. Not14


So looking at this image, the first thing I notice is how much noise digital zoom has. Then I notice that all the bands are the same length which is a good start, so the earlier gel must have just been a fluke. Also this time it appears that the plasmid is at 4kb which is what I expected. According to this image, I would say NotI didn't cut the plasmid, unless it does cut, but it looks too similar to the earlier picture of SapI digestion. I don't know. Koch what do you say?

Steve Koch 23:06, 7 January 2010 (EST): My hunch is that both enzymes are cutting, but I'll want to look at the gel by eye with you tomorrow. Also, need somehow to get better gel images. With ethidium, things were very bright with high contrast. Something that can be done by adding more SYBR or using a better filter on camera?
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