Difference between revisions of "Part:BBa K1632022:Design"
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===Materials and Methods=== | ===Materials and Methods=== | ||
+ | <b>1. Construction</b><br> | ||
+ | 何か一言書く<br> | ||
+ | A. Pcon_lasR_TT_Plux_CmRssrA (6A1) + Plac_rhlI (3K3)<br> | ||
+ | B. Pcon_lasR_TT_Plux_CmRssrA (6A1) + promoter less_rhlI (3K3)<br> | ||
+ | |||
+ | [[Image:|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br> | ||
+ | |||
+ | <b>2. Assay protocol</b><br> | ||
+ | 1.Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.<br> | ||
+ | 2.Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.<br> | ||
+ | 3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br> | ||
+ | 4.Suspend the pellet in 1 mL of LB containing Amp and Kan.<br> | ||
+ | 5.Add 30 microL of suspension in the following medium.<br> | ||
+ | a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)<br> | ||
+ | b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 99.5% ethanol (3 microL)<br> | ||
+ | c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)<br> | ||
+ | 6.Grow the samples of cells at 37°C for more than 8 hours.<br> | ||
+ | 7.Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br> | ||
===Source=== | ===Source=== |
Revision as of 13:17, 13 September 2015
J23100_rbs_lasR_TT_Plux_rbs_CmRssrA
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 383
Illegal AgeI site found at 580 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 927
Design Notes
sequence confirmed
Materials and Methods
1. Construction
何か一言書く
A. Pcon_lasR_TT_Plux_CmRssrA (6A1) + Plac_rhlI (3K3)
B. Pcon_lasR_TT_Plux_CmRssrA (6A1) + promoter less_rhlI (3K3)
[[Image:|thumb|center|600px|Fig. 1. Plasmids]]
2. Assay protocol
1.Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2.Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4.Suspend the pellet in 1 mL of LB containing Amp and Kan.
5.Add 30 microL of suspension in the following medium.
a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 99.5% ethanol (3 microL)
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)
6.Grow the samples of cells at 37°C for more than 8 hours.
7.Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)
Source
Composite of BBa_K553003, BBa_K1632021