Jessica Karen Wong/Notebook/2007-6-21

To Do

 * Get Jason to log in to point mutate I2054
 * Make plan for next week
 * Digest backbones, J04650, B0032, R0040
 * PCR T9002, E0240

Digest

 * See protocol
 * Digested plasmids 1AT3, 1AK3, and 1AC3 with EcoR1 and Pst
 * Used 3ul of AT3 and AC3 plasmid and 4ul AK3 to insure 1 ug of DNA
 * Used EcoR1 buffer
 * Digested B0032 and R0040 with EcoR1 and Spe
 * Used 2ul of B0032 DNA 3ul of R0040 DNA
 * Used EcoR1 buffer
 * Digested J04650 with Xba and Pst
 * Used 5ul of prepped DNA
 * Used buffer 3 as suggested by NEB for the XbaI/PstI digests
 * Incubated at 37C overnight

PCR

 * Did a 100ul preparatory PCR on T9002 and E0240
 * Based on Endy:Colony PCR
 * 95.25 ul PCR Mix
 * 25ul Thermo Polymerase Buffer
 * 25ul dNTP's (10x)
 * 3.75ul Rev Primer (40uM)
 * 2.5ul Taq Polymerase
 * 183 Water
 * 1 ul DNA (3x dilution)
 * 3.75 ul forward primer (because T9002 and E0240 have different forward primers but the same reverse primer)
 * Dilute primers to 40 uM
 * Added 730ul water to E0240F
 * Added 821ul to T9002F
 * Added 925ul to E0240R
 * Did a 3x dilution of DNA samples

Sequencing

 * Nishant prepped and sent I0255 to be sequenced