User:Anthony Salvagno/Notebook/Research/2010/01/06/Purification, Sap Cap, and Ligation Cleanup

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Today will be a 4 part process:

Purification of SapClones

Yesterday I noticed that I was getting weird numbers from the nanodrop, and also that the SapClones smelled like Phenol. So to start now I am going to purify the tubes with a good old Qiaquick cleanup. Koch had mentioned that he used to get better yields when heating up the elution buffer, so I emailed Qiagen to ask about this:

Dear Dr. Salvagno (editor's note: I like the formailty), Thank you for contacting QIAGEN. There are 2 PCR purification kits: QIAquick and Minelute, the range recovered is 100 bp to 10 kb and 70 bp to 4 kb, respectively. If you have Minelute you’ll have to warm the elution buffer at 65°C before applying it to the column for elution, but the recovery will still be low if the fragments are a lot bigger than 4 kb. If you have QIAquick there’s no need to warm the buffer. If I can help you in any other way, please feel free to reply to this email or call me at the number below. Sincerely, Person from Qiagen

So as you can see, I do not need to heat the buffer because I use Qiaquick. Thanks Qiagen!

Determination of Clones' Molarity

  • Sap14 - 16.5ng/ul
  • Sap16 - 19.8ng.ul
  • Sap21 - 18.5ng/ul
  • Saplib - 28.0ng/ul

Attachment of SapCap Digestion of Clones

After getting small DNA amounts with the digested shotgun clones, I am deciding to do the 1 STEP over again, this way I can start with more DNA. Also to save on time and effort, I will just do one clone as a test subject before working with all the clones I have. {{#widget:Google Spreadsheet |key=tpmtgco1c2BUv8xoJpm5xVg |width=250 |height=300 }} I guessed at the amount of SapI to put in the tube. I figured that I had about 250ng/ul of DNA in the tube because it came from a miniprep. It turns out there was only 90ng/ul but this might help with the shoddy digestion SapI tends to provide.

Shotgun Clones Concentration

What I am basing my numbers on.
  • D - 327.3ng/ul ~ 61nM-49nM (based on 8kb-10kb guess)
  • 2 - 283.4ng/ul ~ 93nM-84nM (4.5kb-5kb guess)
  • 14 - 89.7ng/ul ~ 33.75nM (4kb guess)
  • 16 - 191ng/ul ~ 52nM-44nM (5.5-6.5kb guess)
  • 21 - 225.1ng/ul ~ 84nM (4kb guess)

It's funny how these names mean almost nothing to me anymore. Legacy is great, although my simply separating XhoI digest from double digest was pretty clever. Also funny is how I managed to pick the one with the least amount of DNA even though it should have probably had the most (based on that image over there).