Difference between revisions of "Team:Toulouse France/Experiments"

All the following protocols were inspired by one or several protocols, used, improved and optimized (which took more or less time...).
Finally they gave us some results :-).

E. coli competent cells : Cacl2 method

• 1. Do an overnight pregrowth of E.coli DH5α in 5mL of LB at 37°C with agitation.
• 2. Measure the absorbance at 600nm.
• 3. In an Erlenmeyer, add in 100mL of LB medium the volume of pregrowth corresponding to an initial absorbance of 0.05. Let at 37°C with agitation until the absorbance reaches 0.5.
• 4. Aliquote in 50mL sterile tubes and put at 4°C for 10 minutes to slow down the metabolism.

From now on, everything has to be done at 4°C!

• 5. Centrifuge 10 minutes at 6000g and discard the flow-through.
• 6. Resuspend gently the pellet without doing bubbles or vortexing in 20% of the culture volume of CaCl2 sterile solution at 100mM.
• 7. Incubate 20 minutes in ice.
• 8. Centrifuge 10 minutes at 6000g and discard the flow-through.
• 9. Resuspend gently in 50% of the culture volume of CaCl2 solution at 100mM and 15% of sterile glycerol.
• 10. Aliquote 200µL in sterile and cold microcentrifuge tubes.
• 11. Store at -80°C.

Cloning mix by digestion/ligation method

Digestion

• 1. For 20µL of reaction:
• 2µL of buffer
• 1µL of each restriction enzyme
• Complete to 20µL with water and DNA (2-4µg or more if it is a small insert).
• 2. Incubate at 37°C 1 hour or less if using fastdigest enzymes (thermofisher).

Inactivation of the restriction enzymes and purification on columnn
A GeneJET Gel Extraction Kit from Thermofisher is necessary for this step.

• 1. Add 1:1 volume of Binding buffer.
• 2. Vortex briefly.
• 3. Transfer up to 800 μL of the mixture to the GeneJET purification column. Centrifuge for 1 minute at 10,000-14,000rpm. Discard the flow-through and place the column back into the same collection tube.
• 4. Add 700 μL of Wash Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
• 5. Centrifuge the empty GeneJET purification column for an additional 1 minute to completely remove residual wash buffer.
• 6. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube and wait 5 minutes to let all the ethanol evaporate.
• 7. Add 30µL of clean water to the center of the purification column membrane. Wait 2 minutes. Centrifuge for 1 minute.
• 8. Discard the GeneJET purification column and store the purified DNA at -20 °C.

Isolation of fragments by electrophoresis and excision
Use this protocol only if the fragment of the digestion results in several fragments larger than 100bp and that only one is of interest.
A GeneJET Gel Extraction Kit from Thermofisher is necessary for this step.

• 1. Do an agarosis gel at 1% of agar in TAE.
• 2. Put 2µL of 1kb ladder
• 3. Fill wheels with digestion (no need for loading dye if Green FastDigest buffer used)
• 4. Migrate for 20-30 minutes at 100V.
• 5. Put in BET for 5 minutes and rinse 5 minutes in water.
• 6. Reveal under high UV light.
• 7. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
• 8. Add 1:1 volume of Binding Buffer to the gel slice (volume: weight).
• 9. Incubate the gel mixture at 50-60 °C for 10 minutes or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column.
• 10. Transfer up to 800 μL of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 minute. Discard the flow-through and place the column back into the same collection tube.
• 11. Add 700 μL of Wash Buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
• 12. Centrifuge the empty GeneJET purification column for an additional 1 minute to completely remove residual wash buffer.
• 13. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube and wait 5 minutes to let all the ethanol evaporate.
• 14. Add 30µL of clean water to the center of the purification column membrane. Wait 2 minutes. Centrifuge for 1 minute.
• 15. Discard the GeneJET purification column and store the purified DNA at -20 °C

Ligation

• 1. For 20µL of reaction:
  Up to 12µL of DNA (molar ratio 1:3 between vector and insert)
  2µL of ligation buffer
  0.5µL of T4 ligase enzyme
  Water to complete the 20µL
• 2. Leave 1 hour at room temperature

Cloning mix with Gibson method

Gibson mix

• 1. 320µL of 5X ISO buffer (25% PEG8000, 500mM Tris-HCl pH7.5, 50mM MgCl2, 50mM DTT, 1mM dNTP, 5mM NAD)
  0.64µL of T5 exonuclease
  20µL of Phusion polymerase
  40µL of Taq ligase
  820µL of water
• 2. Aliquote 160 PCR tubes with 7.5µL of mix.

Gibson assembly

• 1. Add 2.5µL of DNA per tube (molar ratio 2:1 vector/insert) with 100ng of vector
• 2. In thermocycler put 5 minutes at 37°C and 57 minutes at 50°C.

Cloning mix: other method

Mix 25ng of vector with five times more of insert to obtain 2.5µL.

Transformation

• 1. Defrost the competent cells in ice for 15-20 minutes.
• 2. Resuspend 5 to 10µL of cloning mix in 50µL of cells (or 25µL of cells for the other method) and let in ice for another 20 minutes.
• 3. Do a thermic choc at 42°C for 45 seconds.
• 4. Incubate in ice for 5 minutes.
• 5. Add 500mL of SOC and incubate at 37°C for 1 hour for an ampicillin resistance or 2 hours for chloramphenicol and kanamycin resistance.
• 6. Centrifuge 1 minute at 8000g.
• 7. Discard the flow-through but let 100mL of medium.
• 8. Resuspend the cells.
• 9. Spread on a LB plate with the corresponding antibiotic.

Plasmid extraction

A GeneJET Plasmid Miniprep Kit from Thermofisher is needed for this step.

• 1. Put a colony in 5mL of LB with the corresponding antibiotic to grow overnight.`
• 2. Centrifuge the culture and discard the flow-through.
• 3. Resuspend the pelleted cells in 250 μL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube.
• 4. Add 250 μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
• 5. Add 350 μL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
• 6. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
• 7. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
• 8. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
• 9. dd 500 μL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
• 10. Repeat the wash procedure (step 9) using 500 μL of the Wash Solution.
• 11. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution.
• 12. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube and wait 5 minutes to let all the ethanol evaporate.
• 13. Add 30µL of clean water to the center of the purification column membrane. Wait 2 minutes. Centrifuge for 1 minute.
• 14. Discard the GeneJET purification column and store the purified DNA at -20 °C.

PCR

• 1. For a final volume of 50µL:
  31µL of water
  2.5µL of forward primer
  2.5µL of reverse primer
  1µL of template
  1.5µL of DMSO
  1µL of dNTPs
  10µL of HF buffer
  0.5µL of Phusion polymerase
• 2. Thermocylcer conditions (save for some exception with primers larger than 25bp):
  95°C, 5 minutes
  95°C, 30 seconds
  55°C, 30 seconds
  72°C, 30 seconds - 1 minute/kb

Repeat the last 3 steps 30 times
72°C 5 minutes
Hold 4°C.

PCR fusion

• 1. Same mix as a classic PCR with 50ng of several templates with 40bp of overlaps.
  The primers used are the one the furthest extremities.
• 2. Thermocycler conditions are the same that for a classic PCR but the elongation time must be calculated for the fused fragment.

PCR on colony

• 1. For a final volume of 25µL:
  9µL of water
  12.5µL of dreamtaq mastermix
  1.25µL of forward primer
  1.25µL of reverse primer
  1µL of template
• 2. Thermocycler conditions:
  94°C, 4 minutes
  94°C, 30 seconds
  TM, 20 seconds
  72°C, 1kb/minute

Repeat the last 3 steps 30 times
72°C, 10 min
Hold 4°C.

B. subtilis transformation

Four solutions are necessary before starting the transformation.

Solution 1: Tri-Na Citrate 300 mM

• 0.88 g of Tri-Na Citrate
• 10 mL of mQ water

Wrap in aluminium foil and store at -20°C.

Solution 2: Ferric NH4 citrate

• 0.22 g of Ferric NH4
• 10 mL of mQ water

Wrap in aluminium foil and store at -20°C.

Solution 3: Competence Medium (MC 10X)
For a final volume of 100 mL, you will need:

• 14.04 g of K2HPO4
• 5.24 g of KH2PO4
• 20 g of glucose
• 10 mL of Tri-Na Citrate 300 mM (solution 1)
• 1 mL of Ferric NH4 citrate (solution 2)
• 1 g of Casein Hydrolysate
• 2 g of Potassium Glutamate

The complete mixture should be dissolved in 100 mL. First add 50 mL of milliQ water and mix. When everything is dissolved, add mQ water until 100 mL. Filter sterilize the complete mixture and store at -20°C.

Solution 4: Competence medium (MC 1X)

• 1.8 mL de mQ water
• 200 µL of 10X MC (solution 3)(filter sterilized)
• 6.7 µL of MgSO4 1M autoclaved
• 10 µL of Tryptophan 1% filter sterilized (stored in aluminium foil)

The day before the transformation, collect the Bacillus subtilis strain and drop it in 5 mL of liquid LB. Then grow overnight at 37°C.

1- In a tube containing 2 mL of completed MC (1X), add the volume necessary of B. subtilis culture to reach a OD of 0.04.
2- Grow at 37°C for 5 hours, which should corresponds to the end of the exponential phase of the growth cells.
3- Mix 400 µL of culture with DNA in a fresh tube of 15 mL, loosely closed to ensure the aeration. Usually add 1 µL of DNA, or 10 µL of Qiagen plasmid miniprep, or < I µL of chromosomal prep.
4- Grow the cells at 37°C for an additional 2 hours.
5- Centrifuge the tube at 8 000 RCF during 3 minutes.
6- Empty the supernatant until 100 µL left, and resuspend the pellet in it. Then spread these 100 µL reaction mix on selective antibiotic plates, and incubate at 37°C overnight.


The transformation of Bacillus subtilis can also start after 6 hours of incubation instead of 5 hours. It depends of the end of the exponential phase of the culture.