• 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

• 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

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

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

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

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

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

• 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

• There must be 3x as much insert as vector
• Total volume of solution must be 20ul
• Negative control contains no inserts

Content	ul
Nuclease-free water	Bring to volume
10X T4 Ligase Buffer	2
T4 Ligase	1
Vector : Insert Ratio	1:3