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Protocols

Making cells competent

Transformations

Standard transformation procedure

• Remove competent cells from -80, let thaw for 10 min on ice and aliquot in 50 ul amounts.
• add 2-5 ul of vector, usually in H2O, to 50 ul cells, no mixing by pipet due to shear induction.
• keep on ice for 20 minutes (vector spreading through volume)
• heat shock (42°C) for 45 seconds
• keep on ice for 2 minutes
• add 200 ul SOC, put on 37°C for 1 hour or longer with agitation.
• plate out 250 ul on appropriate antibiotics.

Restrictions

Try to do a restriction in a relatively large volume. As a rule of thumb, use a volume of 50 ul / 500 ng DNA.

• Calculate the amount of DNA you want to use
• add H2O
• add 10 x H buffer (Roche)
• add your calculated amount of DNA
• add 0.5 ul of each enzyme. Keep in mind 0.5 ul = 5 U, where 1 U is defined as the amount of enzyme cutting 1000 ng of DNA / hour, so for extremely large amounts of DNA adjust this.
• keep on 37°C for 2-3 hours.

Ligation

First make sure you have purified the DNA after restriction. Ligation should be in a small volume (we usually use 15 ul), so elute your DNA from the column in a small volume/high concentration.

• add H2O
• add 10 x ligation buffer
• add backbone and insert (theoretically in a 1:3 or 1:4 ratio, for 3A assembly it seemed to work at 1:1 ratios, possibly even better). DNA amounts added are at least 50 ng of the backbone and if possible 100-150 ng of the insert DNA (including it's backbone).
• add 1 ul of T4 Ligase.
• keep the reaction at 16ºC for at least 2 hours, but o/n is preferable.
• if used for transformation, all DNA can be added to competent cells, or if you want to analyze it on gel, keep 5 ul.

PCR

Colony PCR

• Make biobrick mastermix, containing per sample:
  • 12.5 ul Taq mastermix
  • 2.5 ul 10x forward biobrick primer
  • 2.5 ul 10x reverse biobrick primer
  • 7.5 ul H2O
• Put 25 ul in the PCR tubes.
• With a toothpick or pipet point, touch a colony and stir it through the fluid
• Run the iGEM colpcr program (to be added later)

DNA gels

• Take a flask of 0.8% up to 1.5% molten agarose from the 70oC stove.
• Pour a it in a taped gel tray.
• Add ca. 5 ul of SYBRSafe (depending on size gel)
• Add a comb and let the gel harden for ca. 15 minutes.
• Remove the comb and the tape and put the gel tray in an electrophoresis tray.
• Add enough 1x TBE to completely cover the gel.
• Add DNA loading buffer to your samples and load them.
• Let the gel run at a voltage between 60V and 120V, depending on desired resolution/time available.
• Visualize the DNA by putting it in the imager for taking a picture, or if you want to cut out your DNA, put it on the blue light emitter.

Luciferase Assays

Due to some protocols not working as desired, we've used various different ones. The one listed here is the latest, others can be found under the following links: 25th of September protocol; 15th of October protocol

Buffers