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To do

  • Miniprep J04500 to get high volumes
  • Midiprep J04500
  • Digest J04500 /SP @ 2h @45 and KaiC /XP @ 2h @45
  • CIP treat J04500 /SP
  • Gel and purify both
  • Vacufudge both to ~8uL total
  • Ligation/transformation

Midiprep J04500

Perry kindly volunteered to do the midiprep.

Sequencing of Sample "A", "E", "I", and "M" from yesterday

Used primer VR for each, expected ~500bp. Will get results tomorrow, submitted with Perry's stuff.

Digest 4500/SP and KaiC /XP

4 samples of each digested for 2h @ 45C. Run on gel; image pending.

Click for legend

Gel extraction and Post Digest CIP

Gel extracted the first two KaiC segments with each other and the each 4500/SP seperately; got around 30 ng/uL for the 4500/SP in buffer EB. CIP'd the 4 J4500 segments for 1h@37 using 1uL AP.

PCR Purification of 4 J4500 CIP products

PCR purified all 4 J4500 CIP products in one contaner; got 110ng/uL eluting in 30uL water, for around a 80% recovery.

Vacufuge C1

Vacufuged sample "C1" in order to further concentrate DNA, from 30uL to around 5uL. This will be an interesting test to see what the EB salts will do to our ligation product... I'm using as much DNA as I can and not worrying about salt content to see exactly how bad Roche warnings are.