IGEM:MIT/2007/Notebook/2007-8-17: Difference between revisions
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#Plate transformants | #Plate transformants | ||
#Send mer operon PCR products (merR and Pmer) to sequence | #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''' |
Revision as of 11:34, 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