IGEM:MIT/2006/Notebook/2006-7-21
From OpenWetWare
Jump to navigationJump to search
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.
