Team:Buenos Aires/ protocols

From 2013.igem.org

(Difference between revisions)
(Created page with "<div id="external"> = MINIPREP = ==Materials== * Resuspension buffer * Lysis buffer * Neutralization buffer * Isopropanol * 70% Etanol * dH2O * Ice (in ice bucket/container) ...")
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# Let the tube open until it is dried. The pellet must turn from white to transparent.
# Let the tube open until it is dried. The pellet must turn from white to transparent.
# Add 20 ul of dH2O and freeze.
# Add 20 ul of dH2O and freeze.
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=Transformation=
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We have E. Coli DH5α strain competent bacteria, with a transformation efficiency of 10
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'''Important!''' Competent bacteria should always be on ice.
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* If the plasmid comes from the kit or is a product of ligation, add 2μl of plasmidic DNA into 50μl
 +
 +
of competent bacteria. If it’s a plasmid that comes from a miniprep, use a mass in function of
 +
 +
the efficiency of the bacteria.
 +
 +
* Incubate for 20 min on ice.
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 +
* Heat Shock: 42°C for exactly 1’30’’, then immediately place on ice.
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* Incubate for 5 min on ice.
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* Recovery: Add 950μl of LB without antibiotic, incubate from 30 min to an hour at 37°C
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* Pellet the bacteria by centrigue at 10000rpm for 5 minutes. Discard the supernatant of LB.
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Resuspend the bacteria in the remnant LB.
 +
 +
* Plaque onto plates with LB and the desired antibiotic.
 +
 +
* Incubate overnight in a stove at 37°C
 +
 +
 +
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Revision as of 23:01, 22 September 2013

Contents

MINIPREP

Materials

  • Resuspension buffer
  • Lysis buffer
  • Neutralization buffer
  • Isopropanol
  • 70% Etanol
  • dH2O
  • Ice (in ice bucket/container)

Procedure

  1. Centrifuge for 5 minutes at 5000 rpm 1,5 ml of bacteria culture.
  2. Take the pellet and add 250 ul of resuspension buffer and pipet up and down a few times.(make sure that all is properly mixed)
  3. Add 250 ul of Lysis buffer and invert the tubes 5 times to mix. Let rest 5 minutes. The mix must turn transparent.
  4. Add 250 ul of cold Neutralization buffer and invert the tubes 5 times to mix. The mix must turn turbid.
  5. Centrifuge at 13.000 rpm for 15 minutes.
  6. Transfer the supernatant to a new clean eppendorf tube.
  7. Add isopropanol (the same volume that is in the tube). Mix many times and put it in ice for 20 minutes.
  8. Centrifuge at 13.000 rpm for 20 minutes.
  9. Eliminate the supernatant (be carefull that the pellet does not go with it)
  10. Add 1 ml of etanol 70% and use a vortex to ensure the pellet mixes with the etanol.
  11. Centrifuge at 13.000 rpm for 5 minutes.
  12. Eliminate all the supernatant with a pipette (be carefull that the pellet does not go with it)
  13. Let the tube open until it is dried. The pellet must turn from white to transparent.
  14. Add 20 ul of dH2O and freeze.


Transformation

We have E. Coli DH5α strain competent bacteria, with a transformation efficiency of 10

Important! Competent bacteria should always be on ice.

  • If the plasmid comes from the kit or is a product of ligation, add 2μl of plasmidic DNA into 50μl

of competent bacteria. If it’s a plasmid that comes from a miniprep, use a mass in function of

the efficiency of the bacteria.

  • Incubate for 20 min on ice.
  • Heat Shock: 42°C for exactly 1’30’’, then immediately place on ice.
  • Incubate for 5 min on ice.
  • Recovery: Add 950μl of LB without antibiotic, incubate from 30 min to an hour at 37°C
  • Pellet the bacteria by centrigue at 10000rpm for 5 minutes. Discard the supernatant of LB.

Resuspend the bacteria in the remnant LB.

  • Plaque onto plates with LB and the desired antibiotic.
  • Incubate overnight in a stove at 37°C


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