IGEM:MIT/2006/Notebook/2006-7-14

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Contents

TO DO

Andre: re-do SPP PCR (followed by PCR cleanup & gel); find out about exponential phase promoter (make liquid culture for miniprep if possible), research other smells

Stephen: Site-directed mutagenesis of ATF1 + gel; Make indole knockout cells competent (to be followed by transforming Dudareva plasmid); re-do SPP PCR (followed by PCR cleanup & gel), make liquid culture of coding region + terminator transformants, make RBS cultures

Veena: GC on different BSMT concentrations and indole knockout, order primers for BAT2, THI3, pCHBA

Indole Knockout Strain

The indole knockout strain was made competent using the following protocol:

http://openwetware.org/wiki/Preparing_chemically_competent_cells

Site-directed mutagenesis for ATF1

Redone using the Strategene manual available here: http://openwetware.org/wiki/Endy:Site-directed_Mutagenesis

We got the primers ready for mutagenesis using the following protocol:

http://openwetware.org/wiki/PNK_Treatment_of_DNA_Ends

Liquid cultures

We got many, many transformants hopefully carrying SAMT connected to B0015 and BSMT connected to B0015. We made 4 liquid cultures from each type of colony. We also made two liquid cultures from the glycerol stock of cells containing the ribosome binding sites B0030 and B0032. I plan to come in tomorrow to miniprep each of these 10 liquid cultures for eventual sequencing (in the case of the SAMT.B0015 and BSMT.B0015 cultures) and ligation (for each culture).

GC

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