IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-23

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Redo of yesterday's procedure

CIP of digested 011/sp

1h@37C

gel of post-CIP'd

1h15@130V. 1.2%

  • 1: 1kb+
  • 2: undigested 11
  • 3: empty
  • 4: 11/sp marker
  • 5: 11/sp (not shown)
  • 6: 11/sp (not shown)
  • 7: 11/sp (not shown)
  • 8: 11/sp (not shown)
  • 9: 11/sp marker
  • 10: 1kb+

post-stain

Very carcinogenic :D 40uL EtBr, 20min

see above for legend
verification correct band cutout

gel purification and pcr purification

2000mg gel; purified into 6 tubes. sent all 6 tubes into 1 pcr purification. eluted in 30 eb.

ligation

  • J36011 /SP + J36010 /XP (latest), respectively.
    • BB vector: 70ng @ (70 ng/uL) = 1 uL
    • BB insert: 3*(~1800/3700bp)*70ng=102.16 ng @ (20.2 ng/uL) = 5.06 uL
    • 5x dilution: 2uL
    • h20: 1.94uL

sequencing

Sent 4 orders: 11 Vf2&vr and 22 vf2&vr, rush order.

Transformation

Transformants plated at 8:00 PM, placed in incubator at 8:25 PM.

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