Difference between revisions of "Team:Stony Brook/Experiments"

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<li> Amounts of reagents listed below </li>
 
<li> Amounts of reagents listed below </li>

Revision as of 18:00, 22 July 2016

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Protocols & Experiments

Universal Lab Protocols

Agarose Gel Preparation

  • Add 50mL of 1X TAE buffer to a 500mL erlenmeyer flask
  • Add 0.5g of 1% agarose
  • Microwave for three 20 second intervals and cool slightly
  • Add 1ul Ethidium Bromide
  • Mix and pour into gel box. Add Comb
  • Cool for 30 minutes

YPD Media

  • For liquid media
    • Add 20g of Bacto peptone, 10g of yeast extract and 950mL of water to a flask
    • Autoclave on the liquid setting
    • Add 50mL of 40% glucose and mix
  • For solid media
    • Add same reagents as liquid, plus 24g of Bacto agar
    • Autoclave
    • Stir on a magnetic stir plate and add 50ml of 40% glucose
    • Pour into petri dishes

LB Broth

Add items to flask:

  • 5g Tryptone
  • 2.5g Yeast Extract
  • 5g NaCl
  • H2O filled to 500mL
  • Mix with magnetic stir bar on low heat, autoclave for liquids



Nanodrop

  • Select Nucleic Acid
  • Clean, drop 1ul water, measure
  • Blank
  • Measure with 1ul of substance
  • For PCRs: 260/280 ratio should be near 2

LB Agar

Add items to flask:

  • 5g Tryptone
  • 2.5g Yeast Extract
  • 5g NaCl
  • 7.5g Agar
  • H2O filled to 500mL
  • Mix with magnetic stir bar on low heat, autoclave for liquids



    • NEB Monarch Nucleic Acid Purification Protocols

    The following NEB kits were used and protocols can be found on their website

    • Monarch Plasmid Miniprep Kit
    • Monarch DNA Gel Extraction Kit
    • Monarch PCR & DNA Cleanup Kit

Digestion

  • Add Nuclease free water to a 2ml test tube
  • Add 10X buffer for restriction enzymes
  • Add restriction Enzymes
  • Add DNA using (DNA needed in ng/concentration in ug/ul) = ul
  • Make negative controls without the enzymes
    • Make up volume with nuclease free water
  • Amounts of reagents listed below

    • 50ul Reaction

      Content ul
      Nuclease-free water Bring to volume
      10X Buffer 5
      Restriction Enzymes 1 each
      DNA Calculate volume for 2ug DNA

    10ul Reaction

      Content ul
      Nuclease-free water Bring to volume
      10X Buffer 5
      Restriction Enzymes .5 each
      DNA Calculate volume for 2ug DNA

    Transformation

    • Thaw competent E. coli cells on ice and pipette 50ul cells into a transformation tube
    • Add 1-5ul containing 1pg-100ng of plasmid DNA and flick tube 4-5 times
    • Place tube on ice for 30 minutes
    • Heat shock at 42°C for 30 seconds
    • Place on ice for 5 minutes
    • Add 950ul room temperature SOC to mixture
    • Incubate at 37°C for 60 minutes at 250rpm
    • Mix and perform several 10-fold serial dilutions in SOC
    • Spread 50-100ul onto a plate and incubate overnight at 37°C

    Cancer Group Protocols


    Vaccine Group Protocols

    Inspiration