IGEM:MIT/2007/Notebook/2007-8-17: Difference between revisions
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Polystyrene binding | Polystyrene binding | ||
working | 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 | *'''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 | |||
seemed fine with 1ppb to 1ppm | seemed fine with 1ppb to 1ppm | ||
*'''look up icp-aes works | *'''look up icp-aes works | ||
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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? |
Latest revision as of 11:36, 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