IGEM:MIT/2007/Notebook/2007-8-17

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(Agenda)
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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 14:34, 17 August 2007

Agenda

  1. Transform R0051 into BL21
  2. Transform last night's ligations (F+B, I.B34.CPX.B14) into BL21
  3. Plate transformants
  4. 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
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