"
Team:Hawaii/Antibiotic test for BB-pRL1383a
From 2008.igem.org
(Difference between revisions)
(→Transformation) |
(→Transformation) |
||
| Line 29: | Line 29: | ||
# Cells were heat shocked at 42C for 1 minute, then returned to ice for 2 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. | # 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 | + | # Cells were incubated at 37C with shaking at 250 rpm for 70 minutes. |
# 50 μl of transformed cells + SOC were plated LB+sp<sub>variable</sub>. 20 μl of untransformed cells + SOC were plated on control plates. | # 50 μl of transformed cells + SOC were plated LB+sp<sub>variable</sub>. 20 μl of untransformed cells + SOC were plated on control plates. | ||
# Plates were incubated at 37C for 2 days. | # Plates were incubated at 37C for 2 days. | ||
Revision as of 03:12, 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 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.
- Plates were incubated at 37C for 2 days.
