User:Barry Canton/Notebook/T7 RNAP transcription of rRNA/2008/07/30

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  • 1E9 cells in the extract maybe
  • That means 1E9*1E13 copies of the 16s rRNA
  • That is 1E13/6E23 or 1/6 E-10 moles of 16s sites
  • That is 2E-11 moles
  • In 10μl that is 20μM
  • I planned on making them up to 100μM
  • To get this, I added 281μl or 209μl respectively to the primers

Labeled wt probe for about 2.5 hrs and then left on ice for about 2.5 hrs. I took the TOP10 extract and did 1:2 dilutions into DEPC water. I added 5μl of the extraction and 5 dilutions onto fresh membrane. I crosslinked for 5 min at high power on the big Sauer lab UV transilluminator. I prepared fresh hybridization buffer and incubated buffer plus membrane at 62C for a few minutes, I then added all the prepared probe and put at 30C in a roller. Will leave overnight.