IGEM:MIT/2006/Notebook/2006-7-21

Indole Knockout
Made liquid cultures of other knockout strains

Annealed osmY primers
Followed the procedure here http://openwetware.org/wiki/Endy:Annealing_complementary_primers

osmY PCR
Done with each pair of new primers meant for PCRing both the short and long osmY promoter region.

Digest
R0040

5 uL Buffer 2

.5 uL BSA

32 uL 14 ng/uL DNA

.5 uL PstI

.5 uL SpeI

B0030 & B0032

33.5 uL H20

5 uL Buffer 2

.5 uL BSA

10 uL 75 ng/uL DNA

.5 uL PstI

.5 uL XbaI

Ligation
4.5 uL H20

1 uL T4 DNA Ligase Buffer

.5 uL T4 DNA Ligase

2 uL Stationary Phase Promoter (Annealed primers)

2 uL AMP Backbone


 * Low Concentrations of cut R0040, B0030, and B0032 found. Low concentration of the short PCRed stationary phase promoter found.  Good concentrations of long PCRed stationary phase promoter and annealed stationary phase promoter primers found.

Smell Test
Did not work for any of the cultures. Sequencing in progress.