Team:TUDelft/Protocols

From 2008.igem.org

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==Luciferase Assays==
==Luciferase Assays==
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===Cell preparation===
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We've used multiple protocols, but some didn't work reliably. The latest protocol is below here, others are listed behind the links: [[25th of September protocol]]
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*Pellet 1 ml of grown culture when the culture is around 0.6 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge.
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*Lyse the cells by adding 1x lysis buffer from the Renilla Luciferase Assay kit.
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*Freeze the cells at -20 until measurements will be done.
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===Measurements===
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*Mix 1 ul of 100X luciferase substrate in 100 ul of assay buffer per sample in the luminometer's reagent tube no. 1
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*Put 20 ul of lysed cells in a white 96 wells plate.
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*Put the prime plate in the luminometer (usually on top)
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*Put the tube with assay buffer under reagent needle no. 1, make sure the tip of the needle is in a position to reach all the assay buffer.
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*Prime the luminometer with 1000 ul assay buffer in the priming plate(make sure the tubing is rinsed before)
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*Measure luciferase activity by:  
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**Adding 100 ul of assay buffer to a well
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**Wait 2 seconds
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**Integrate luminescence for 10 seconds.
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**Repeat for every well
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**There is a standard protocol on the computer in which you only have to indicate the wells to be assayed.
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*Purge the tubing (Assay buffer can be stored and frozen for short periods (1 week at most) according to the technical manual)
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*Rinse the tubing with 5000 ul of ethanol, and purge it.
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*Rinse the tubing with 5000 ul of H2O, and purge it.
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*The tubing and injector should be clean and empty now.
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==Protein gels==
==Protein gels==

Revision as of 12:14, 20 October 2008

Contents

Protocols

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, 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 better)
  • add 1 ul of T4 Ligase.


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

We've used multiple protocols, but some didn't work reliably. The latest protocol is below here, others are listed behind the links: 25th of September protocol

Protein gels

RNA extraction

Buffers

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