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

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Current revision (14:36, 17 August 2007) (view source)
 
Line 9: Line 9:
Polystyrene binding
Polystyrene binding
working
working
-
-replacing AHL inducible promorter with constutive promoter
+
*replacing AHL inducible promorter with constutive promoter
-
-ran a western, presented results
+
*ran a western, presented results
-
-increasing AHL, more protein (issues loading); thinking put in set amounts of known protein
+
*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
+
**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
*'''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)
Line 25: Line 25:
order hg standards samples
order hg standards samples
$30/hr to use
$30/hr to use
-
-might run komasi stain if expression of lpp-ompa to find concentration which is iptg inducible
+
*might run komasi stain if expression of lpp-ompa to find concentration which is iptg inducible
seemed fine with 1ppb to 1ppm
seemed fine with 1ppb to 1ppm
*'''look up icp-aes works
*'''look up icp-aes works
Line 31: Line 31:
Mercury Sensor
Mercury Sensor
-
3A of I13500, mer operon, iat3 - failed
+
#3A of I13500, mer operon, iat3 - failed
-
mercury binding assay - failed
+
#mercury binding assay - failed
-
pcr -first pcr worked, second pcr with same primers failed (shouldn't work acc. to tk)
+
#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
+
#gel - failed 600 bp in pcr product and - control, then new one no bands
*'''annealing temp?
*'''annealing temp?

Current revision

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

  1. 3A of I13500, mer operon, iat3 - failed
  2. mercury binding assay - failed
  3. pcr -first pcr worked, second pcr with same primers failed (shouldn't work acc. to tk)
  4. 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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