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

 * 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