Julius B. Lucks/Antisense RNA Transcription Attenuation/Brantl Wagner Recapitulation: Difference between revisions
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* see [[Beta-Galactosidase_Assay_(A_better_Miller)]] for a Miller assay protocol | * see [[Beta-Galactosidase_Assay_(A_better_Miller)]] for a Miller assay protocol | ||
** can we do this on plates? - see [[LacZ_staining_of_cells]] and [[Knight:Beta-galactosidase_assay/96_well_format]] | ** can we do this on plates? - see [[LacZ_staining_of_cells]] and [[Knight:Beta-galactosidase_assay/96_well_format]] | ||
** we need to ask around for anyone that has done Miller assays | |||
** we might also consider making the plasmids with GFL instead of lacZ | |||
== Verifying Table 2 == | == Verifying Table 2 == |
Revision as of 21:58, 31 January 2008
Goals
- To verify that we can grow the plasmids given to us by Brantl.
- To verify that we can re-create the transcriptional attenuation results (Table 2 of Sabine Brantl, E Gerhart H Wagner J Bacteriol (2002) vol. 184 (10) pp. 2740-7).
- in-vivo beta-galactosidase reaction
- RTPCR
Growing Plasmids
- Transform plasmids into Tg1 cells
- mini-prep
- test-digest
- sequence relevant regions
- If doesn't work in Tg1 cells, then try in DH10B
Verifying transcriptional and translational fusions work
- grow cells on plates that will trigger lacZ
- start by making a constitutively active lacZ protein inside a cell and growing on a plate - maybe have a biobricked lacZ?
- should all have positive hits
- this will be a practice that we can do library screening with lacZ
- see Beta-Galactosidase_Assay_(A_better_Miller) for a Miller assay protocol
- can we do this on plates? - see LacZ_staining_of_cells and Knight:Beta-galactosidase_assay/96_well_format
- we need to ask around for anyone that has done Miller assays
- we might also consider making the plasmids with GFL instead of lacZ
Verifying Table 2
- do double transformations according to the combinations in Table 2
- when plate up, observe the look of colonies that have functional antisense gene
- is the attenuation observable?
- we might have to make constitutively active versions of pUCpIII and pMGI
- How do we perform the quantitative beta-galactosidase experiment?
- do we get similar attenuation factors
RTPCR verification
- how do we go about doing this? Should we try to perform their equivalent of Figure 3? How do we do this with constitutively active anti-sense RNAs? Do we do a pulse of inducer, and then freeze transcription?
- this would be good to practice to measure how any library hits measure to the native version.