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  1. Make frozen stocks of the KaiA+bkb and KaiB+bkb transformants that we grew up in liquid culture yesterday
  2. Miniprep the above
  3. (Maybe) Do an X-P digest of the KaiA+bkb and KaiB+bkb plasmids (though we may want to wait and do this along with the other KaiA+bkb and KaiB+bkb ligation transformants) (Will do later) completed 8/7/06
  4. Inoculate the KaiA+bkb and KaiB+bkb transformants from yesterday's ligation
  5. Add CIP to the E-S digest of LC vector
  6. Run the digests from yesterday on a gel (E-S digest of LC vector, X-P digest of J04450+bkb)
  7. Gel-extract and purify the LC vector and J04450 insert if they exist (Will purify tomorrow, along with the high-copy backbone that the J04450 was inserted in)
  8. (maybe) Perform a ligation and transformation of the J04450 insert into LC vector (Will do after we gel-purify them) completed 8/7/06
  9. Do 12 minipreps for Mingming
  10. Check sequencing on the Genewiz website (the orders have not been processed)

KaiA/B + bkb inoculation

We found 10 colonies on the KaiA+bkb plate and 5 colonies on the KaiB+bkb plate. We inoculated all 15 colonies in liquid culture (8 mL LB, 8 uL Amp) and placed them in the 37C shaker around 1830.

Gel images of E-S digest of LC vector and X-P digest of J04450+bkb (high copy)

Click for legend
Click for legend

We extracted the high-copy vector (2kb) and J04450 insert (1kb) from lanes 2 and 4, and the low-copy vector (3.5 kb) from lanes 6 and 8.