User:Anthony Salvagno/Notebook/Research/2009/03/11/Digestion, Ligation, Transformation Retry Day 1

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I want to be ambitious and get all the way up to the transformation part today, but that will really only save me one day. Anyways right now I am digesting pRS413 DNA and gDNA with XhoI.

The Digestion

I am following the same procedure as last time, exactly.

Gel Prep
Tube Number Vol pRS413 Vol gDNA Vol and Type 10x Buffer Vol BSA 10x Vol XhoI Vol H2O Total Vol
1 33.5uL ---- NE2/8.3uL 8.3uL 3.3 29.5uL 82.5uL
2 ---- 23.4uL NE2/7.5uL 7.5uL 3.75uL 32.75uL 74.9uL

This time we are not doing the gel stuff, this is because the last time it worked out well and since we are doing the exact same prep nothing should change.
Steps:

  1. Mix components as demonstrated in chart (make sure to add XhoI last)
  2. Allow 2 hours for reaction
  3. Add more XhoI to tube 2 and CIP to tube 1
  4. Allow 1 hora mas
  5. Phenol Extraction of both
  6. EtOH precipitate 1 hour
  7. Resuspend in 25uL 1X TE buffer
  8. Get nanodrop reading
  9. Prepare for Ligation

Notes:

My lab notebook rocks. I've used it so many times to easily search for protocols and information I can't quite remember and need to lookup. I love it. Thanks OWW!