User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/24

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Check overnight growth

Good!

I checked overnight growth of nine 10ml bottles with kanamycin and selected colonies.

  1. 4 bottles based on pUA66katE#A1#2, a verified colony that contains the katE insert
  2. 3 bottles from separate plates with colonies containing SAP treated vectors that have been ligased
  3. 2 bottle with a standard ligase

My question was - whether the SAP treatment ensured the presence of my insert given that the colonies grew?

I proceeded using 2 bottles of pUA66katE#A1#2 for miniprep, I kept one to preform assay and the last bottle was centrifuged and the cell pellets frozen. For the SAP treated vectors, 2 were used for miniprep. For the standard ligation 1 bottle was used for miniprep. All remaining bottles were centrifuged and the cell pellets frozen at -20ºC.

Glycerol stock

I decided to do a miniprep on 4 of 8 bottles:

  1. pAU66katE#A1#2-1 (known to contain the insert)
  2. pAU66katE#A1#2-2 (known to contain the insert)
  3. pUA66katE#A1, SAP
  4. pUA66katE#B1, SAP
  5. pUA66katE#2, Normal Ligase

In order to preserve any sample that screened positive for the insert, I prepared glycerol stock.

For each of the 5 samples:

  1. 700µl bacteria sample
  2. 300µl of 50% Glycerol
  3. Place in -80ºC freezer

Miniprep

Digestion