Team:Reading/Protocols

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A note on protocols

Contents

Below are a number of examples of protocols - modified and non-modified - used throughout our project. The protocols we list below may not have been followed exactly in each instance of usage, often due to time constraints - beginning an experiment on one day and concluding it on another.


  1. A Note on Protocols
  2. The Goods
  3. Acknowledgements
  4. References

The Goods

Here are all of the protocols.

PCR

PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.

Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage.

2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix [add reference here?].

The PCR program used was as follows:

Start:
  • 95C/30s

    • 35 cycles:
  • 95C/10s
  • 56C/15s
  • 72C/70s

    • Final:
  • 72C/5min
  • 68C/10min

    • All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol1.


    Potassium ferricyanide assay

    Another potential protocol


    Isolation of plasmid DNA from bacteria (miniprep)

    1. Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes
    2. Discard supernatant by pipetting off ensuring not to disturb the pellet
    3. Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes
    4. Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times
    5. Centrifuge at 13,000 rpm for 5 minutes
    6. Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate
    7. Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm
    8. Discard the flow through
    9. Add 500 µl of wash solution to the column
    10. Centrifuge for 1 minute at 13,000 rpm
    11. Discard flow through
    12. Add 500 µl of wash solution to the column
    13. Centrifuge for 1 minute at 13,000 rpm
    14. Discard flow through
    15. Centrifuge for 1 minute at 13,000 rpm
    16. Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube
    17. Add 35 µl of ultrapure water. Do not touch the membrane with the pipette
    18. Incubate at room temperature for 2 minutes
    19. Centrifuge for 2 minutes at 13,000 rpm


    Glycerol cell stock generation

    This protocol is adapted from 2 freely available protocolsREFERENCE, REFERENCE. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet

    1. Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips
    2. Centrifuge at 13,000 rpm for 2 minutes
    3. Discard supernatant
    4. Add 0.5 ml of 60% glycerol stock
      1. 240 ml of glycerol
      2. 160 ml nano pure water
      3. mix together and autoclave
        1. freeze at -80℃


          E. coli transformation

          Another potential protocol


          Synechocystis transformation

          Another potential protocol

    References

    1. http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf

    Acknowledgements

    Everyone we need to thank for help with protocols.

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    rusynbioigem@gmail.com

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