IGEM:MIT/2007/Notebook/2007-8-17: Difference between revisions
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--discussion about t7 and its 2 bands, uncertainty why 2 bands | --discussion about t7 and its 2 bands, uncertainty why 2 bands | ||
--could be multimers (perhaps not a worry if it's working) could be modification of the protein | --could be multimers (perhaps not a worry if it's working) could be modification of the protein | ||
''' | *'''double check t7 control | ||
*have to put stop codon translational stops (could be read through) | *'''have to put stop codon translational stops (could be read through) | ||
*do finer material in bulk | *'''do finer material in bulk | ||
Revision as of 11:35, 17 August 2007
Agenda
- Transform R0051 into BL21
- Transform last night's ligations (F+B, I.B34.CPX.B14) into BL21
- Plate transformants
- Send mer operon PCR products (merR and Pmer) to sequence
Meeting Notes
Polystyrene binding working -replacing AHL inducible promorter with constutive promoter -ran a western, presented results -increasing AHL, more protein (issues loading); thinking put in set amounts of known protein --discussion about t7 and its 2 bands, uncertainty why 2 bands --could be multimers (perhaps not a worry if it's working) could be modification of the protein
- double check t7 control
- have to put stop codon translational stops (could be read through)
- do finer material in bulk
Mercury Binding
received ec20 in mail with lppompa
determining hg2+, mass spec won't work
use icp-aes instead with half day training session
order hg standards samples
$30/hr to use
-might run komasi stain if expression of lpp-ompa to find concentration which is iptg inducible
seemed fine with 1ppb to 1ppm
- look up icp-aes works
Mercury Sensor
3A of I13500, mer operon, iat3 - failed
mercury binding assay - failed
pcr -first pcr worked, second pcr with same primers failed (shouldn't work acc. to tk)
gel - failed 600 bp in pcr product and - control, then new one no bands
- annealing temp?
- try again. negative control should be no template
Review of overall project
Things need to be completed -replace iptg operon by mer operon so hg inducible -mer operon mission critical
- many of e coli dna remnants are in lab equipment (like primers, etc) might not even need plasmid
- needs crisp benchwork
- possible parallel efforts or drag in grads etc