Team:Stony Brook/Protocols

Protocols & Experiments



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, press okay
  • Blank with 1ul
  • Measure with 1ul of substance
  • 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

Digest

  • Add Nuclease free water to a 2ml test tube
  • Add 10X buffer for restriction enzymes
  • Add restriction Enzymes
  • Add DNA using (Need 500ng of DNA for 25ul)
  • 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 DNA

10ul Reaction

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

Ligation

Content ul
Nuclease-free water Bring to volume
10X T4 Ligase Buffer 2
T4 Ligase 1
Vector : Insert Ratio 1:3
  • There must be 3x as much insert as vector
  • Total volume of solution must be 20ul
  • Negative control contains no inserts

Phosphatase

  1. Add 1pmol of DNA ends
  2. Add 2ul of AP Buffer
Content ul
AP Buffer 2
Phosphatase 1 ul
Plasmid 10
H2O 7