Team:TUDelft/Protocols

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(Difference between revisions)
(→Protein gels)
(→Luciferase Assays)
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==Luciferase Assays==
==Luciferase Assays==
 +
===Cell preparation===
 +
*Pellet 1 ml of grown culture when the culture is around 0.6 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge.
 +
*Lyse the cells by adding 1x lysis buffer from the Renilla Luciferase Assay kit.
 +
*Freeze the cells at -20 until measurements will be done.
 +
===Measurements===
 +
*Mix 1 ul of 100X luciferase substrate in 100 ul of assay buffer per sample in the luminometer's reagent tube no. 1
 +
*Put 20 ul of lysed cells in a white 96 wells plate.
 +
*Put the prime plate in the luminometer (usually on top)
 +
*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.
 +
*Prime the luminometer with 1000 ul assay buffer in the priming plate(make sure the tubing is rinsed before)
 +
*Measure luciferase activity by:
 +
**Adding 100 ul of assay buffer to a well
 +
**Wait 2 seconds
 +
**Integrate luminescence for 10 seconds.
 +
**Repeat for every well
 +
**There is a standard protocol on the computer in which you only have to indicate the wells to be assayed.
 +
*Purge the tubing (Assay buffer can be stored and frozen for short periods (1 week at most) according to the technical manual)
 +
*Rinse the tubing with 5000 ul of ethanol, and purge it.
 +
*Rinse the tubing with 5000 ul of H2O, and purge it.
 +
*The tubing and injector should be clean and empty now.
==Protein gels==
==Protein gels==

Revision as of 11:51, 23 September 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

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

Cell preparation

  • Pellet 1 ml of grown culture when the culture is around 0.6 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge.
  • Lyse the cells by adding 1x lysis buffer from the Renilla Luciferase Assay kit.
  • Freeze the cells at -20 until measurements will be done.

Measurements

  • Mix 1 ul of 100X luciferase substrate in 100 ul of assay buffer per sample in the luminometer's reagent tube no. 1
  • Put 20 ul of lysed cells in a white 96 wells plate.
  • Put the prime plate in the luminometer (usually on top)
  • 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.
  • Prime the luminometer with 1000 ul assay buffer in the priming plate(make sure the tubing is rinsed before)
  • Measure luciferase activity by:
    • Adding 100 ul of assay buffer to a well
    • Wait 2 seconds
    • Integrate luminescence for 10 seconds.
    • Repeat for every well
    • There is a standard protocol on the computer in which you only have to indicate the wells to be assayed.
  • Purge the tubing (Assay buffer can be stored and frozen for short periods (1 week at most) according to the technical manual)
  • Rinse the tubing with 5000 ul of ethanol, and purge it.
  • Rinse the tubing with 5000 ul of H2O, and purge it.
  • The tubing and injector should be clean and empty now.

Protein gels

RNA extraction

Buffers

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