Team:Stony Brook/Experiments

iGEM SBU 2021

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Protocol for PCR


  • Q5 2X Master Mix: 12.5 uL
  • 10 uM For Primer: 1.25 uL
  • 10 uM Rev Primer: 1.25 uL
  • DNA: 2.5 uL
  • Nuclease free water: 7.5 uL
  • Thermocycler
  • PCR Tubes

Preparing the thermocycler:

  • Denaturation: 98 ℃; 5-10 s
  • Annealing: 70 ℃; 10-30 s
  • Extension: 72 ℃; 90 s (About 30 cycles)
  • Final extension: 72 ℃, 180 s
  • Nuclease free water: 7.5 uL
Step Directions
1 Start the annealing temperature at the higher Tm, go down 1 degree at a time for 1 cycle each until hit (lower Tm-5) and then back cycle that for 20 cycles. Start with 180s initial denaturation at 98 °C and end with 180s final extension at 72 °C.

Protocol for Agarose Gel Electrophoresis:


  • Agarose
  • Gel Tray
  • Power Source
  • Comb
  • 1x TAE
  • Ethidium Bromide
Step Directions
1 Weigh 0.5g agarose powder and 50ml 1X TAE buffer and add them to a flask.
2 Melt the mixture in a microwave until the solution becomes clear, and then add and mix EB or SYBR safe with the solution after the it cools to about 50~60 °C.
3 Pour the solution into the gel casting tray with appreciate comb.
4 Pull out the comb when the gel is completely solid.
5 Place the gel in the electrophoresis chamber and add enough TAE Buffer to submerge the gel.
6 Pipette DNA samples mixed with appreciate amount of loading buffer into wells on the gel.
7 Run the gel at 100V for about an hour.

Protocol for HiFi Assembly


  • Ice
  • DNA vector
  • DNA fragment
  • Pipette andTips
  • Thermocycler
  • NEB HiFi DNA Assembly Master Mix
Step Directions
1 Set up the following reaction on ice, according to NEB:
2 Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
3 Transform NEB DH5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μl of the chilled assembled product, following the transformation protocol.

Protocol for NanoDrop


  • NanoDrop
  • Pipette
  • Pipette Tips
  • Tubes with prepared DNA
  • Kimwipes
Step Directions
1 Make a blank measurement by pipetting 1µl of water onto the NanoDrop and press blank on the NanoDrop program.
2 Wipe the droplet of water
3 Pipette 1 µl of DNA solution, close the NanoDrop and press measure.
4 Record and label the results.
5 Repeat for all DNA samples.

Protocol for Competent Cells


  • LB Media
  • Agar plate
  • Pipette
  • Sterile 50 ml tube
  • Centrifuge
  • MgCl2
Step Directions
1 Streak out the E. coli strain on an LB plate to isolate colonies and incubate at 37 °C overnight.
2 Use a sterile pipette tip to collect cells from a single colony and inoculate in 5 ml sterile liquid LB Medium at 37 °C overnight in a shaker incubator.
3 Pipette 1 mL cells to 100 mL liquid LB Medium at 37 ℃ shaker incubator until grow the cultures to OD600 reaching about 0.26.
4 Pipette 25 ml of the cells to a pre-chilled sterile 50 ml tube.
5 Use a centrifuge to rotate at 4 °C, 4000 rpm for 10 minutes.
6 Decant supernatant and resuspend the cells by 10 ml ice cold 100 mM MgCl2. Hold on ice for 30 minutes.

Protocol for E. coli Transformation


  • Competent Cells
  • DNA plasmid
  • Heat block and ice
  • SOC media or LB media
  • Agar Plates
Step Directions
1 Take competent cells out of -80°C and thaw on ice.
2 Warm up agar plates warm up to 37°C in the incubator.
3 Add of 2 μL vector into 50 μL competent cells, and GENTLY mix by flicking the bottom of the tube.
4 Incubate on ice for 20 min.
5 Heat shock each transformation tube in the 42°C water bath for 30 s.
6 Put the tubes back on ice for 2 min.
7 Add 950 μL SOC media or LB media(without antibiotic) to the bacteria and grow in a 37°C shaking incubator for 60 min.
8 Plate the transformation and incubate plates at 37°C overnight.

Protocol for Protein expression of BL21(DE3) using IPTG


  • Transformed BL21(DE3) bacteria in LB media
  • Pipette and Tips
  • 100 mM stock solution of IPTG
Step Directions
1 The NEB protocol was followed.

Protocol PP1 Assay

Step Directions
1 Protocol from Moore et al. paper was followed.