Team:Tartu TUIT/Experiments

Restriction Digest

20 minutes (Incubation time is not considered)
  • Water to 50μL
  • Buffer (NEB or FastDigest Green Buffer (10X)) 5 μL
  • DNA Template up to 5 μL
  • Restriction enzymes 2.5 µL of each restriction enzyme
  • FastAP 1.25 µL
  • Thermostate (37oC)
  • Cold body (To keep restriction enzymes)
  1. Keep all reagents on ice.
  2. Make a reaction mix into 20 µL or 50 µL (depends on your restriction) volume in a microfuge tube.
  3. Add reagents in following order: water, buffer (NEB or FastDigest Green Buffer (10X)), DNA template, restriction enzyme, FastAP (if recommended by your supervisor).
  4. Mix gently and spin down.
  5. Incubate at 37oC in a thermostate for 1 hour or overnight (overnight for NEB Buffer & at maximum 1 hour for FastDigest Green Buffer (10X)).
  6. Analyze your results via gel electrophoresis.
  1. https://www.genscript.com/restriction-digestion-protocol.html
  2. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012556_FastDigest_ApaI_UG.pdf

PCR Purification

20 minutes
  • FADF Buffer
  • Wash Buffer
  • Elution Buffer
  • Heat block (55oC)
  • Vortex
  • FADF Column
  • Collection Tube
  • Centrifuge Machine
  1. Excise the agarose gel with a clean scalpel.Remove the extra agarose gel to minimize the size of gel slice.
  2. Transfer up to 300 mg of the gel slice into a microcentrifuge tube.The maximum volume of gel slice is 300 mg.
  3. Add 500 µL of FADF Buffer to the sample and mix by vortexing.For > 2% agarose gel, add 1000 µL of FADF Buffer.
  4. Incubate at 55oC for 5-10 minutes and vortex the tube every 2~3 minutes until the gel slice dissolved completely.During incubation, interval vortexing can accelerate the gel dissolved.Make sure that the gel slice has been completely dissolved before proceed the next step.After gel dissolved, make sure that the color of sample mixture is yellow. If the color is violet, add 10 µL of sodium acetate, 3M, pH 5.0. Mix well to make color of sample mixture turned to yellow.
  5. Cool down the sample mixture to room temperature. And place FADF Column into a Collection tube.
  6. Add 800 µL of sample mixture to the FADF Column. Centrifuge at 11,000 x g for 30 seconds, discard the flow-thorugh. If the sample mixture is more than 800 µL, repeat this step for rest of sample mixture.
  7. Add 750 µL of Wash Buffer (ethanol added) to the FADF Column. Centrifuge at 11,000 x g for 30 seconds and discard the flow-through.Make sure that the ethanol (96 - 100%) has been added to Wash Buffer when first use.
  8. Centrifuge again at full speed (18,000 x g) for an additional 3 minutes to dry the column matrix.Important step! The residual liquid should be removed thoroughly on this step.
  9. Place the FADF Column to a new microcentrifuge tube.
  10. Add 40 µL of Elution Buffer or ddH2O to the membrane center of the FADF Column. Stand the FADF Column for 1 min. Important step! For effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.Important: Do not elute the DNA using less than suggested volume (40 µL). It will lower final yield.
  11. Centrifuge at full speed (~ 18,000 x g) for 1 min to elute DNA.

DNA Concentration Measurement

5-10 miutes
  • Water
  • Sample
  • Elution Buffer
  • NanoDrop Microvolume Spectrophotometer
  1. Choose Nucleic Acid from the menu
  2. Open the sample arm. Clean the flat surfaces from both sides.
  3. Pipette around 1~2 µL of water to the sample surface. Click Ok and machine will be ready to work.
  4. Add around 1~2 µL of Elution Buffer to make Blank measurement. Click “Blank”. Clean the surfaces from both sides.
  5. Pipette around 1~2 µL of sample to sample surface. Click “Measurement”. Now, sample result is ready.

Agarose Gel preparation

1 hour
  • TAE Buffer
  • Agarose
  • Ethidium Bromide
  • Conical flask
  • Gel caster
  • Comb
  1. Add 100 mL of TAE Buffer to conical flask
  2. Weigh 2 grams of agarose and add to 100 mL of buffer solution (2% agarose gel)
  3. Keep in oven until agarose is completely melted
  4. Take the solution from oven
  5. Add ethidium bromide (4 µL of ethidium bromide per 100 mL of TAE Buffer)
  6. Poor the solution to a gel cluster
  7. Place the comb
  1. http://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=2

Bacterial Transformation

1 hour 30 minutes – 2 hours (Incubation time is not considered)
  • Strain E.coli DH5a
  • Plasmid DNA
  • LB liquid medium
  • Ice
  • Selection plates
  • hermostate 37oC
  • Heat block (42oC)
  1. Take the competent cells out of -80C fridge and put them on ice and let them melt
  2. Take 1 µL of plasmid DNA and put it in a separately labelled eppendorf
  3. Add 50 µL of the cells, resuspend gently
  4. TLeave it on ice for 30 minutes
  5. Add ethidium bromide (4 µL of ethidium bromide per 100 mL of TAE Buffer)
  6. Poor the solution to a gel cluster
  7. Heat shock at 42oC for 2 minutes
  8. Put them on ice for 2 minutes
  9. Add 400 µL of LB medium into the eppendorfs, put them all in a flask and shake at 37oC for 30 minutes
  10. Take selection plates from cold room and leave them on the table (label them and add around 10 glass beads to each plate)
  11. Take eppendorfs out and centrifuge them at 6000 rpm for 60 seconds
  12. Remove supernatant and resuspend cells in the remaining liquid
  13. Pipette cells onto the selection plates and shake them until surface is dry
  14. Transfer the glass beads into the bid container containing ethanol
  15. Let cells grow at 37oC overnight in thermostate
  1. http://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=2

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