Team:Reading/Protocols

From 2014.igem.org

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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 Thermo Scientific NanoDrop machine. All PCR tubes were kept on ice prior to usage.  
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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.  
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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 mastermix [add reference here?].
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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?].
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The PCR program used was as follows:
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<b>Start:</b>
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<li> 95C/30s </li>
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<b>35 cycles:</b>
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<li> 95C/10s </li>
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<li> 56C/15s </li>
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<li> 72C/70s </li>
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<b>Final:</b>
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<li> 72C/5min </li>
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<li> 68C/10min </li>
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All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit [reference here too?].
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<!-- Potassium ferricyanide assay -->
<!-- Potassium ferricyanide assay -->

Revision as of 15:56, 13 October 2014

University of Reading
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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 [reference here too?].


    Potassium ferricyanide assay

    Another potential protocol


    E. coli transformation

    Another potential protocol


    Synechocystis transformation

    Another potential protocol

    References

    Here are the references.

    Acknowledgements

    Everyone we need to thank for help with protocols.

    Facebook
    Twitter
    rusynbioigem@gmail.com

    Retrieved from "http://2014.igem.org/Team:Reading/Protocols"