Jessica Karen Wong/Notebook/2007-7-5

To Do

• PCR clean and then Digest T9002 with Nsi1
• Run gel of I2056
• Drop off sequencing

T9002

• Heat Shocked Mfe1 digest
• PCR cleaned, DNA concentration was only 19 ng/ul
• Continued the sequential digest
  • Used all the Mfe1 digest product (30ul), 12.5 ul water, buffer 3
• Also trying a double digest of Mfe1 and Nsi1 in buffer 2
  • 11ul scarred DNA, 31.5 ul water
• PCR cleaned both
  • Sequential Digest DNA concentration 10.9 ng/ul
  • Double Digest DNA concentration 19 ng/ul

I2056

• Ran gel of the overnight colony PCR
• Have a faint band of the right size
• Overnighted 2 cultures
  • One for glycerol and sequencing, one to miniprep for scarring
  • Used 5ul Amp and 10ul Tet per 5ml culture

E0240

Gel from 7/5/07

• Did a 100ul preparatory PCR at 53.5
• Fwd primer BB_E0240_F was 38.2nmole added 956ul water
• Rev primer BB_backbone was 38.09nmole added 952ul water
• Ran a gel of the PCR, sample to the right of 2 log ladder
  • Faint band of correct size (3kb) but much brighter band that was too small (1kb)
• Didn't use a long enough elongation time
• Will retry with longer elongation time and both vent and phusion polymerases