Team:Hawaii/Antibiotic test for BB-pRL1383a
From 2008.igem.org
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# Plates were incubated at 37C for 19 hours. | # Plates were incubated at 37C for 19 hours. | ||
- | + | ==Results== | |
+ | {|class=wikitable border=1 align=center | ||
+ | !Plate | ||
+ | !Cells | ||
+ | !Colonies | ||
+ | !Blue? | ||
+ | |- | ||
+ | |align=center|LB | ||
+ | |align=center|untransformed DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|no | ||
+ | |- | ||
+ | |align=center|LB+X-gal | ||
+ | |align=center|untransformed DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB | ||
+ | |align=center|untransformed DH5α | ||
+ | |align=center|0 | ||
+ | |align=center|no | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>2.5</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>5</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>10</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>15</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>20</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>25</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|lawn | ||
+ | |align=center|yes | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>40</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|56 large, too many to count pinpoint, smear present | ||
+ | |align=center|no | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>50</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|29 large, too many to count pinpoint, smear present | ||
+ | |align=center|no | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>75</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|41 large, ~30 pinpoint | ||
+ | |align=center|no | ||
+ | |- | ||
+ | |align=center|LB+sp<sub>100</sub> | ||
+ | |align=center|J33207+pRL1383a ligation in DH5α | ||
+ | |align=center|75 large, 12 small | ||
+ | |align=center|no | ||
+ | |} | ||
+ | |||
===Discussion=== | ===Discussion=== |
Revision as of 23:37, 22 October 2008
Contents |
Antibiotic test and Blue/White screen for BB-pRL1383a
Objective
- To verify the successful construction of BB-pRL1383a, a pRL1383a derivative in BioBrick format with blue/white screening capabilities
- To determine the ideal concentration of spectinomycin for isolation of E. coli colonies transformed with BB-pRL1383a
- To verify the ability to use blue/white screening for selection of transformed cells and to determine length of incubation necessary for this screen
Materials and Methods
Media
To test for antibiotic selection, 30 ml LB + 1.5% agar plates were made. Spectinomycin were added at the following concentrations:
- sp2.5
- sp5
- sp10
- sp15
- sp20
- sp25
- sp40
- sp50
- sp75
- sp100
Thirty microliters of 20X X-gal (20 mg/ml) was added to each plate to a final concentration of 1 μg/ml.
Control plates, also 30 ml LB + 1.5% agar, were as follows:
- LB only
- + X-gal (1 μg/ml)
- + sp100
Transformation
- 100 μl of competent DH5α cells were thawed on ice for 10 minutes.
- 5 μl of a [http://partsregistry.org/Part:BBa_J33207 J33207] and pRL1383a ligation reaction were added to the cells. Ten microliters of cells were left untransformed.
- Cells were incubated on ice for 5 minutes.
- Cells were heat shocked at 42C for 1 minute, then returned to ice for 2 minutes.
- 400 μl SOC were added to the transformed cell mixture and 50 μl were added to the untransformed aliquot.
- Cells were incubated at 37C with shaking at 250 rpm for 70 minutes.
- 50 μl of transformed cells + SOC were plated LB+spvariable. 20 μl of untransformed cells + SOC were plated on control plates.
- Oops. 100 μl was plated on LB+sp<sub.100</sub> plate.
- Plates were incubated at 37C for 19 hours.
Results
Plate | Cells | Colonies | Blue? |
---|---|---|---|
LB | untransformed DH5α | lawn | no |
LB+X-gal | untransformed DH5α | lawn | yes |
LB | untransformed DH5α | 0 | no |
LB+sp2.5 | J33207+pRL1383a ligation in DH5α | lawn | yes |
LB+sp5 | J33207+pRL1383a ligation in DH5α | lawn | yes |
LB+sp10 | J33207+pRL1383a ligation in DH5α | lawn | yes |
LB+sp15 | J33207+pRL1383a ligation in DH5α | lawn | yes |
LB+sp20 | J33207+pRL1383a ligation in DH5α | lawn | yes |
LB+sp25 | J33207+pRL1383a ligation in DH5α | lawn | yes |
LB+sp40 | J33207+pRL1383a ligation in DH5α | 56 large, too many to count pinpoint, smear present | no |
LB+sp50 | J33207+pRL1383a ligation in DH5α | 29 large, too many to count pinpoint, smear present | no |
LB+sp75 | J33207+pRL1383a ligation in DH5α | 41 large, ~30 pinpoint | no |
LB+sp100 | J33207+pRL1383a ligation in DH5α | 75 large, 12 small | no |