Team:Hawaii/Antibiotic test for BB-pRL1383a

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# 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.
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# Cells were incubated at 37C with shaking at 250 rpm for 1 hour.
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# Cells were incubated at 37C with shaking at 250 rpm for 70 minutes.
# 50 &mu;l of transformed cells + SOC were plated LB+sp<sub>variable</sub>. 20 &mu;l of untransformed cells + SOC were plated on control plates.
# 50 &mu;l of transformed cells + SOC were plated LB+sp<sub>variable</sub>. 20 &mu;l 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

  1. To verify the successful construction of BB-pRL1383a, a pRL1383a derivative in BioBrick format with blue/white screening capabilities
  2. To determine the ideal concentration of spectinomycin for isolation of E. coli colonies transformed with BB-pRL1383a
  3. 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

  1. 100 μl of competent DH5α cells were thawed on ice for 10 minutes.
  2. 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.
  3. Cells were incubated on ice for 5 minutes.
  4. Cells were heat shocked at 42C for 1 minute, then returned to ice for 2 minutes.
  5. 400 μl SOC were added to the transformed cell mixture and 50 μl were added to the untransformed aliquot.
  6. Cells were incubated at 37C with shaking at 250 rpm for 70 minutes.
  7. 50 μl of transformed cells + SOC were plated LB+spvariable. 20 μl of untransformed cells + SOC were plated on control plates.
  8. Plates were incubated at 37C for 2 days.

Results

Discussion

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