IGEM:MIT/2007/Notebook/2007-8-7: Difference between revisions
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==Agenda== | |||
# Transform last night's ligation into DH5a | |||
# Plate | |||
# Group Meeting at 2 in 674 | |||
===Transformed Ligation product of Standard Assemblies (I14032+B0034, R0051+B0034, CPX+B0014)=== | ===Transformed Ligation product of Standard Assemblies (I14032+B0034, R0051+B0034, CPX+B0014)=== | ||
Plated 150µl and spun down remainder, centrifuged, resuspended concentrated pellet and plated that as well | Plated 150µl and spun down remainder, centrifuged, resuspended concentrated pellet and plated that as well (pellet was small) | ||
===Streaked CPX BL21 cells for Second Western Blot=== | ===Streaked CPX BL21 cells for Second Western Blot=== | ||
| Line 20: | Line 25: | ||
*need to do negative control of (no plasmid) | *need to do negative control of (no plasmid) | ||
*check specificity and try polypropylene etc | *check specificity and try polypropylene etc | ||
*arrange in order of increasing concentrations | *in future, arrange results in order of increasing concentrations | ||
*do viable count of bacteria at high concentrations | *do viable count of bacteria at high concentrations (ask grads about this) | ||
T7 fluorescent antibody tag | |||
*scale bar | *scale bar for pictures | ||
*bright+fluorescent | *bright+fluorescent | ||
Xis it same scale across all images or does program chooses intensity. eric answered this | Xis it same scale across all images or does program chooses intensity. eric answered this | ||
* | *Future controls: "induce" cells without plasmid. stain with only second antibody. | ||
*hi res of stained antibody-can we tell localized binding (to each other) or to polystyrene? | |||
*hi res of stained antibody-can we tell localized binding or to polystyrene? | |||
what's next | what's next: | ||
we've done in pbs, but need to do in water | we've done in pbs, but need to do in water | ||
need to put cpx downstream of promoter | need to put cpx downstream of promoter | ||
| Line 59: | Line 63: | ||
*do chlorampheticol amplification on plasmid | *do chlorampheticol amplification on plasmid | ||
grow overnight, dilute, regrow in chlorampheticol; inhibits protein production but not dna replication->all of energy goes into copying; 10x yield on low copy plasmids. (must check if not chloramphenicol resistant) | grow overnight, dilute, regrow in chlorampheticol; inhibits protein production but not dna replication->all of energy goes into copying; 10x yield on low copy plasmids. (must check if not chloramphenicol resistant) | ||
*use TE instead of water | *use TE instead of water (trys-EDTA, trys is a buffer, EDTA chelates magnesium, a cofactor of DNase) | ||
*can heat it; if heat elution buffer to 50 or 60 degrees, | *can heat it; if heat elution buffer to 50 or 60 degrees, | ||
*elute by 30 ul then combine | *elute by 30 ul twice, then combine | ||
Revision as of 13:01, 7 August 2007
Agenda
- Transform last night's ligation into DH5a
- Plate
- Group Meeting at 2 in 674
Transformed Ligation product of Standard Assemblies (I14032+B0034, R0051+B0034, CPX+B0014)
Plated 150µl and spun down remainder, centrifuged, resuspended concentrated pellet and plated that as well (pellet was small)
Streaked CPX BL21 cells for Second Western Blot
Things we need to do are starred
8-7 Minutes update Talked about polystyrene binding-works Mercury binding-ordered mercury sensing-not working compare random sequence to e coli genome then make part from it talked about polystyrene assay *need to do negative control of (no plasmid) *check specificity and try polypropylene etc *in future, arrange results in order of increasing concentrations *do viable count of bacteria at high concentrations (ask grads about this) T7 fluorescent antibody tag *scale bar for pictures *bright+fluorescent Xis it same scale across all images or does program chooses intensity. eric answered this *Future controls: "induce" cells without plasmid. stain with only second antibody. *hi res of stained antibody-can we tell localized binding (to each other) or to polystyrene? what's next: we've done in pbs, but need to do in water need to put cpx downstream of promoter metal binding using ec20 on lpp-ompa *find out how well iron binds to ec20 *find out toxic levels for similar metals (iron, zinc). do background check on this Mike Bennie:<<ask him biobricking beta c term domain of n terminus ~1 week away; most pieces are done. MTA with de Lorenzo. protein export tag fos gune?? leucine zipper beta portion of 84 ege1 has protein autotransporter *Possible leads on psb3k3 purifications: *let tk know; can watch *do chlorampheticol amplification on plasmid grow overnight, dilute, regrow in chlorampheticol; inhibits protein production but not dna replication->all of energy goes into copying; 10x yield on low copy plasmids. (must check if not chloramphenicol resistant) *use TE instead of water (trys-EDTA, trys is a buffer, EDTA chelates magnesium, a cofactor of DNase) *can heat it; if heat elution buffer to 50 or 60 degrees, *elute by 30 ul twice, then combine *what percentage of bacteria is binding? *try fine particulate polystyrene **engineering fish to prevent mercury acculumation **nitrocellulose sandwiches *robbie's a pro at this stuff robbie or rana for western blot thurs 10 am 4-261
