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