Team:Siberia/Experiments

Protocols

Here are not only protocols, but tips we consider being useful for the lab work. They may help the beginners, but even experienced people could find some of them interesting

Agarose gel electrophoresis (non-sterile conditions)

  1. Measure 0.5 g (samples > 1000 bp), or 0.75g (200 bp < samples <1000 bp) of agarose
  2. Prepare at least 350 ul of 1x TAE with Ethidium Bromide at final conc ~ 0.3μg/mL (MUTAGEN! WEAR GLOVES AND LAB COAT)
    3. Tip: It will save your time, if you prepare larger volume of 1x TAE with Ethidium Bromide once and than will use it
  3. Mix it with 50 ml 1x TAE and boil until agarose is melted.
  4. Let the gel cool down to 50 °C, until you'll feel comfortable to touch it with your hand
  5. Pour the agarose into a gel tray with the well comb in place. Wait until it is completely solid (~ 20 min at room temp)
  6. Place the gel into the gel box and cover it with 1x TAE from step 2
  7. Carefully load your samples into the wells. Mix it with loading buffer if needed
    8. Tip: You can use the inner sterile side of the Parafilm slide to mix samples with buffer
  8. Turn on the voltage. You can vary the parameters to achieve better speed or resolution but the defaults are 10d V, 200 mA, where d is the distance between the electrodes
  9. Check the migration rate watching the loading buffer
  10. After the process ends visualize the DNA/RNA using any source of UV-light (wear glasses)

Ethanol DNA precipitation (non-sterile conditions)

  1. Add 0.1 V of 3-5 M NaCL or NaOAc to your sample
  2. Add 2.5-3 V of 96% ethanol
  3. Vortex and put at - 80 °C for 30 minutes
  4. Centrifuge 13 kg, 10 min, 4 °C (temperature influences the DNA amount greatly)
  5. Carefully remove ALL the supernatant (don't disturb the residue, which could be almost invisible)
  6. Let the residual ethanol to evaporate incubating samples in open tubes at 5 min, 37 °C
  7. Dilute your DNA in sterile water (mQ if needed) and store at - 20 °C
    8. Tip: The protocol will help to clear your DNA from salts, prepare it for the electroporation, increase the concentration etc. But it will reduce the total amount of the DNA

Blunt ends ligation tips (non-sterile conditions)

  1. Recommended vector/insertion molar ratio for the process is 1:50
  2. Vector dephosphorylation is highly recommended prior to the ligation. Note, that alkaline phosphatase can show exonuclease activity, if incubated more than written in manufacturer's recommendations
  3. Insertion could be additionally treated with T4 polynucleotide kinase prior to the ligation, for the further increase of the success probability
  4. Don't forget to deactivate the ligase prior to the transformation. 10 min at 65 °C is enough for the T4 DNA ligase. Active ligase can hinder the transformation
  5. To verify the correct orientation with the PCR colony use the vector-specific forward primer and the insertion-specific reverse one

Preparing reagents for the effective heat-shock transformation (non-sterile conditions)

Reagent name Components
MOPS I
  1. 0.1045 g of 3-(N-morpholino)propanesulfonic acid
  2. 0.0603 g of RbCl
  3. 50 ml of dH2O
MOPS II
  1. 1.045 g of 3-(N-morpholino)propanesulfonic acid
  2. 0.0603 g of RbCL
  3. 2.5 ml of 1M CaCl2
  4. 50 ml of dH2O

Filter MOPS reagents through a 0.22 μm filter prior to use and store at 4 ° C.

Competent cells preparation & heat shock transformation (sterile conditions)

  1. Add 50*N μl overnight bacteria suspension to N+1 ml of LB-medium, where N stands for the number of transformations your are going to perform
  2. Incubate with shaking at 37 °C for 2h, then 20 minutes on ice
  3. Transfer the bacteria to N 1.5 ml Eppendorf tubes.
  4. Centrifuge 1 ml of bacterial suspension at 6000 rpm for 4 min at 4 °C
  5. Remove the supernatant
  6. Gently resuspend the bacteria in 1 ml of MOPS I
  7. Repeat the steps 4
  8. Gently resuspend the bacteria in 1 ml of MOPS II
  9. Incubate for 15 min on ice
  10. Repeat the step 4
  11. Resuspend cells in 100 μl of MOPS II
  12. Add the DNA (a total of 50 ng will be optimal)
  13. Incubate 30 min on ice
  14. Heat-shock at 42 °C for 2 min
  15. Add 900 ul of LB medium to each tube
  16. Incubate 1 h at 37 °C. While incubating prepare Petri dishes with LB agar and the desired selective markers
  17. If the DNA you have added at the 12th step was a plasmid, extracted from bacteria, you should only plant 50 μl of bacteria suspension
  18. If the DNA was the result of ligation, plant all cells: centrifuge them at 6000 rpm for 2 minutes and remove 900 μl of supernatant. Plant all bacteria resuspending them with the remaining 100 μl
  19. Incubate the Petri dishes at 37 °C for 14-16 h

Colony PCR (sterile conditions)

  1. Preapare an 0.5 μl Eppendorf tube for each colony you are going to check
  2. Add 20 μl of sterile water to each tube
  3. With a sterile toothpick/pipette tip grab one half of the colony of interest and put it into the water
  4. Heat the tubes at 95 °C for 10 min, then centrifuge at 13000 g for 5 minutes
  5. Use the supernatant as matrix for the PCR reaction with the insertion specific pair of primers

Kits and reagents

Here are all the kits and reagents which we have used according to the manufacturers protocol

Name Purpose Notes (+, if works as expected)
Biolabmix LR HS-ПЦР-Color (2×) High-fidelity PCR +
Sibenzyme Taq-pol with standard buffer Colony PCR +
Evrogen Cleanup Standard gel purification kit Clear the DNA after PCR, restriction digestion, or agarose gel DNA ethanol precepitation is required prior to the restriction, becuse inhibition may occur
Biolabmix Plasmid DNA extraction kit Plasmid DNA extraction Low yield
Sibenzyme T4 DNA ligase Ligation +
Sibenzyme Restriction enzymes: PciSI, AhlI, EcoRV Restriction digestion of the DNA PciSI and AhlI can't be heat-inactivated (manufacturer's information)