Assembly Related Protocols

From 2008.igem.org

(Difference between revisions)
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Transform. (same as usual)
Transform. (same as usual)
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== Transfers from Miniprepped DNA (already in correct vectors - transfers into L/R cells)==
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 +
In 0.5 ml tube, combine:
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*1ul plasmid dna
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*30ul of cells (L/R) + 30 KCM (no water)
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 +
Take the reaction, and:
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*sit in ice for 10 min
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*heat shock for 90 sec
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*sit another 2 min ice
 +
 +
Then, incubate your tubes for 5 minutes (No rescuing is necessary)
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 +
Next, prepare plates (double antibiotics) and tubes with media (LB + antibiotics)
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 +
*Take 15ul of rxn. Spot it onto a plate (no spreading required)
 +
*Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media.
 +
*Let plates and tubes sit overnight.
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*Miniprep the next day.

Revision as of 03:12, 2 August 2008

Newest EcoRI/BamHI Transfer method (7/31/08)

Digestion:

  • 0.5ul plasmid in entry vector
  • 0.5ul EcoRI
  • 0.5ul BamHI
  • 1ul NEB2 + ATP buffer
  • 7.5ul of water
  • incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.

No purification step.

Ligation:

  • 1ul of dilute assembly vector (or 0.5ul if undiluted)
  • 0.5ul ligase

Transform. (same as usual)

Transfers from Miniprepped DNA (already in correct vectors - transfers into L/R cells)

In 0.5 ml tube, combine:

  • 1ul plasmid dna
  • 30ul of cells (L/R) + 30 KCM (no water)

Take the reaction, and:

  • sit in ice for 10 min
  • heat shock for 90 sec
  • sit another 2 min ice

Then, incubate your tubes for 5 minutes (No rescuing is necessary)

Next, prepare plates (double antibiotics) and tubes with media (LB + antibiotics)

  • Take 15ul of rxn. Spot it onto a plate (no spreading required)
  • Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media.
  • Let plates and tubes sit overnight.
  • Miniprep the next day.
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