Team:Heidelberg/pages/Notebook/Methods

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== Make a liquid culture without any plasmid ==
== Make a liquid culture without any plasmid ==
# Inoculate medium
# Inoculate medium

Revision as of 09:53, 14 October 2014

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Contents

Make a liquid culture without any plasmid

  1. Inoculate medium
  2. Grow at the preferred temperature of the organism.

Make a liquid culture carrying three plasmids

  1. Inoculate medium
  2. Grow at the preferred temperature of the organism.

Streak bacteria without any plasmid onto an agar plate

  1. Streak the bacteria onto the agar plate

Dilute non-plasmid dsDNA

  1. Dilute the DNA.

PCA

  1. For a 20 µl reaction, mix equimolar amounts of the DNA fragments (10 ng to 15 ng in total) and add water to a volume of 10 µl.
  2. Add 10 µl 2X Phusion Flash Master Mix.
  3. Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 10 cycles of 1 s denaturation at 98 °C, 5 s annealing at 55 °C, extension at 72 °C for 15 to 30 s/kb of the product; final extension at 72 °C for 5 min.
  4. Add 5 µl of the reaction to 25 µl Phusion Flash Master mix. Add primers to a final concentration of 0.5 µM and water to a final volume of 50 µl.
  5. Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 20 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the product; final extension at 72 °C for 5 min.

DNA purification (Qiagen PCR Purification Kit)

  1. Follow the Qiagen protocol.
  2. Measure the concentration with a NanoDrop.

Make a liquid culture carrying a plasmid

  1. Inoculate medium
  2. Grow at the preferred temperature of the organism.

Dilute plasmid DNA

  1. Dilute the plasmid DNA.

Golden Gate Assembly (isothermal)

  1. Mix 100 ng of the backbone and equimolar amounts of the insert(s) in water for a total volume of 15 µl.
  2. Add 1.5 µl of 10X T4 Ligase Buffer and, when using BsaI or another restriction enzyme that requires it, BSA (Bovine Serum Albumin) at a final concentration of 1X.
  3. Add 1 µl of each restriction enzyme and 1 µl of T4 DNA Ligase (2,000,000 cohesive end ligation units/ml).
  4. Incubate at 37 °C for 1 h, then heat inactivate at 50 °C and 80 °C for 5 min each.

Make competent cells already carrying a plasmid

  1. Do some stuff

Make a glycerol stock of bacteria carrying a plasmid

  1. Mix an aliquot of a LB culture with the same volume of storage buffer
  2. Freeze at -80 °C

Restriction digest (NEB)

  1. Mix purified DNA with water
  2. Add buffer
  3. Add restriction enzyme(s)
  4. Incubate
  5. Optional heat inactivation

Make competent cells not yet carrying any plasmid

  1. Do some stuff

Pick a colony carrying three plasmids from an agar plate

  1. Pick up a sterile tip with a pipette. Dip it into a single colony so it attaches to the tip without applying suction.

Make a liquid culture carrying two plasmids

  1. Inoculate medium
  2. Grow at the preferred temperature of the organism.

Gel Electrophoresis

  1. Measure out 7.5 g of agarose.
  2. Pour agarose powder into microwavable flask along with 500mL of 1xTAE.
  3. Microwave for 1-3min (until the agarose is completely dissolved and there is a nice rolling boil).
  4. Let agarose solution cool down for 5min.
  5. Add 10 µl of 10 mg/l ethidium bromide (EtBr) stock solution (final concentration 0.2 mg/l). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
  6. Pour the agarose into a gel tray with the well comb in place.
  7. Place newly poured gel at 4°C for 10-15 minutes OR let sit at room temperature for 20-30 minutes, until it has completely solidified.
  8. Add loading buffer to each of your digest samples.
  9. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
  10. Fill gel box with 1xTAE until the gel is covered.
  11. Carefully load your samples into the additional wells of the gel (recommended: 15 µl).
  12. Run the gel at 80-150 V until the dye line is approximately 75-80 % of the way down the gel.
  13. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
  14. Using any device that has UV light, visualize your DNA fragments.

PCR from Bacteria (colony PCR or genomic amplification)

  1. Suspend the picked colony in 10 µl to 20 µl of sterile H2O (or use inoculated medium).
  2. Mix 12.5 µl OneTaq 2X Master Mix (NEB) with 1 µl of the bacterial suspension.
  3. Add primers to a final concentration of 0.5 µM.
  4. Add H2O to a final volume of 25 µl.
  5. Run your thermocycler.

Miniprep

  1. Follow the Qiagen protocol
  2. Measure the concentration on a NanoDrop

Heat shock plasmid transformation (1 -> 2 plasmid)

  1. Set a heat block to 42 °C.
  2. Thaw an aliquot chemically competent bacteria on ice.
  3. Add plasmid DNA to the aliquot and mix gently by flicking the tube.
  4. Incubate the bacteria on ice for 10 min to 30 min.
  5. Heat shock the bacteria for exactly 30 s (20 s for E. coli BL21(DE3)).
  6. Incubate the bacteria on ice for approx. 5 min.
  7. Add 1 ml of SOC or LB medium and recover the plasmids for 1 h at 37 °C while shaking (optional if the new plasmid only adds an Ampicillin resistance).
  8. Optionally spin down the recovered suspension and resuspend it in a minimal volume of medium.
  9. Plate the suspension on agar supplemented with the appropriate antibiotics (fill in the appropriate protocol here!).

Plasmid purification (Qiagen PCR Purification Kit)

  1. Follow the Qiagen protocol.
  2. Measure the concentration with a NanoDrop.

Anneal complementary oligonucleotides

  1. Mix equal amounts of the oligonucleotides in an appropriate buffer.
  2. Heat the mixture to 95 °C, then slowly cool it down to room temperature over approx. 45 min (by switching of the heat block or using a ramp-cool method in a thermocycler).

Phusion Flash PCR

  1. Add H2O to a final volume of 50 µl.
  2. Add 25 µl 2X Phusion Flash Master Mix.
  3. Add primers to a final concentration of 0.5 µM.
  4. Add 2.5 pg to 25 ng template DNA.
  5. Run your thermocycler.

Recovery of plasmid DNA from filter paper

  1. Use clean gloves and cut the marked circle area that contains the dried plasmid DNA.
  2. Using clean forceps, insert the filter paper into a 1.5 ml micro centrifuge tube. Add 50 µl of pure water (or until the paper is completely soaked), vortex briefly and incubate at room temperature for 5 minutes. Vortex again and centrifuge the tube for a few seconds.
  3. Remove about 5 µl of supernatant for use in transfecting E. coli by electroporation or chemical means. Please do not try to use the DNA directly for any application other than to transform bacteria and prepare a plasmid stock.
  4. Store the remainder of the filter paper/water mix at -20 or -80 °C as a permanent archive in case that your plasmid stock ever gets lost or if something turns out to be wrong with it.

Streak bacteria carrying three plasmid onto an agar plate

  1. Streak the bacteria onto the agar plate or use sterile glass beads.

Gel extraction of a plasmid

  1. Using a clean scalpel, excise the desired band(s) from the agarose gel.
  2. Follow the Qiagen protocol.
  3. Mesaure the concentration on a NanoDrop.

Dissolve plasmid DNA from the registry distribution

  1. Add 10 µl H2O to the well of the registry distribution if it was not previously used.
  2. Use 1-2 µl for transformation.

Gel extraction

  1. Using a clean scalpel, excise the desired band(s) from the agarose gel.
  2. Follow the Qiagen protocol.
  3. Mesaure the concentration on a NanoDrop.

Heat shock plasmid transformation (2 -> 3 plasmids)

  1. Set a heat block to 42 °C.
  2. Thaw an aliquot chemically competent bacteria on ice.
  3. Add plasmid DNA to the aliquot and mix gently by flicking the tube.
  4. Incubate the bacteria on ice for 10 min to 30 min.
  5. Heat shock the bacteria for exactly 30 s (20 s for E. coli BL21(DE3)).
  6. Incubate the bacteria on ice for approx. 5 min.
  7. Add 1 ml of SOC or LB medium and recover the plasmids for 1 h at 37 °C while shaking (optional if the new plasmid only adds an Ampicillin resistance).
  8. Optionally spin down the recovered suspension and resuspend it in a minimal volume of medium.
  9. Plate the suspension on agar supplemented with the appropriate antibiotics (fill in the appropriate protocol here!).

Streak bacteria carrying two plasmids onto an agar plate

  1. Streak the bacteria onto the agar plate or use sterile glass beads.

Streak bacteria carrying a plasmid onto an agar plate

  1. Streak the bacteria onto the agar plate or use sterile glass beads.

Heat shock plasmid transformation (0 -> 1 plasmid)

  1. Set a heat block to 42 °C.
  2. Thaw an aliquot chemically competent bacteria on ice.
  3. Add plasmid DNA to the aliquot and mix gently by flicking the tube.
  4. Incubate the bacteria on ice for 10 min to 30 min.
  5. Heat shock the bacteria for exactly 30 s (20 s for E. coli BL21(DE3)).
  6. Incubate the bacteria on ice for approx. 5 min.
  7. Add 1 ml of SOC or LB medium and recover the plasmids for 1 h at 37 °C while shaking (optional if the new plasmid only adds an Ampicillin resistance).
  8. Optionally spin down the recovered suspension and resuspend it in a minimal volume of medium.
  9. Plate the suspension on agar supplemented with the appropriate antibiotics (fill in the appropriate protocol here!).

Pick a colony carrying a plasmid from an agar plate

  1. Pick up a sterile tip with a pipette. Dip it into a single colony so it attaches to the tip without applying suction.
  2. Resuspend the colony in sterile medium or water.

Make a glycerol stock of bacteria carrying two plasmid

  1. Mix an aliquot of a LB culture with the same volume of storage buffer.
  2. Freeze at -80 °C.

Make Medium

  1. Put appropriate amount of Mediums powder, put it into correct bottle, label it correctly and put in the correct box for autoclavation

Plasmid ligation with T4 DNA Ligase (NEB)

  1. Mix water and T4 DNA Ligase Buffer (NEB).
  2. Add DNA.
  3. Incubate at room temperature.

Gather bacteria carrying a plasmid from an agar plate

  1. Using a sterile item, gather some bacteria from the plate.

Heat shock plasmid transformation (0 -> 2 plasmids)

  1. Set a heat block to 42 °C.
  2. Thaw an aliquot chemically competent bacteria on ice.
  3. Add plasmid DNA to the aliquot and mix gently by flicking the tube.
  4. Incubate the bacteria on ice for 10 min to 30 min.
  5. Heat shock the bacteria for exactly 30 s (20 s for E. coli BL21(DE3)).
  6. Incubate the bacteria on ice for approx. 5 min.
  7. Add 1 ml of SOC or LB medium and recover the plasmids for 1 h at 37 °C while shaking.
  8. Optionally spin down the recovered suspension and resuspend it in a minimal volume of medium.
  9. Plate the suspension on agar supplemented with the appropriate antibiotics (fill in the appropriate protocol here!).

Heat shock plasmid transformation (1 -> 3 plasmids)

  1. Set a heat block to 42 °C.
  2. Thaw an aliquot chemically competent bacteria on ice.
  3. Add plasmid DNA to the aliquot and mix gently by flicking the tube.
  4. Incubate the bacteria on ice for 10 min to 30 min.
  5. Heat shock the bacteria for exactly 30 s (20 s for E. coli BL21(DE3)).
  6. Incubate the bacteria on ice for approx. 5 min.
  7. Add 1 ml of SOC or LB medium and recover the plasmids for 1 h at 37 °C while shaking.
  8. Optionally spin down the recovered suspension and resuspend it in a minimal volume of medium.
  9. Plate the suspension on agar supplemented with the appropriate antibiotics (fill in the appropriate protocol here!).

Make a glycerol stock of bacteria without any plasmid

  1. Mix an aliquot of a LB culture with the same volume of storage buffer.
  2. Freeze at -80 °C.

Gather bacteria carrying two plasmids from an agar plate

  1. Using a sterile item, gather some bacteria from the plate.

Expression of Dnmt1 (linear)

  1. Transform the plasmid harboring DNMT1 (Kanamycin resistant) into Rosetta DE3 competent cells (Chloramphenicol resistant) ( negative control untransformed)
  2. Inoculate a few colonies to 40 ml LB+Kan+Cm medium to grow the cells overnight at 37 °C (Prepare 4l LB+Kan+Cm medium to autoclave)
  3. Dispense 10 ml of the growth culture into each 1 l flask and grow the cells at 37 °C. When the cell OD reaches ~0.6, lower the temperature to 15-20 °C
  4. 30 min later, add IPTG (0.4 mM final concentration) and ZnCl2 (0.1 mM final concentration) into the media and continue to grow the cells overnight.
  5. Spin down the cells at 4000 rpm for 20 min (4 °C, 5th or 6th floor, fill 500 ml buckets for Ja10 rotor to 375 ml), and resuspend the cell paste in HisTrap loading buffer, with approximately 20 ml buffer for each liter of cell paste. (Take sample S1, accordingly C1 from untransformed control)
  6. SONICATION of the cells (Lohmann group, cold room 4th floor) while keeping them on ice, disrupt the cells using sonication. To avoid the protein degradation and aggregation, add PMSF and DTT to 1 μM and 0.5 mM respectively.
  7. Place the sonication tip in the sample, close to the bottom, but not touching the tube. Select the right program: Microtip: yes; Amplitude 60; Working time 2 min; On 0.02; Off 0.05 After 5 minutes, check if the sample did not heat up and repeat the program one more time. Clean the tip with some ethanol after use.
  8. After the cell is lysed, spin down the cell lysate using high speed or ultracentrifuge (17000 rpm x 45 min, Ti70 rotor, thick wall tubes [355631], filled to 15 ml)
  9. Collect the supernatant. If any aggregates or particles are visible, filter the supernatant with a 0.2 µm filter using a syringe. (Take sample from supernatant S2 and pellet S3, accordingly C2 and C3 from untransformed control)
  10. Load the supernatant to pre-equilibrated HisTrap column, wash the column using 200 ml of chilled His trap loading buffer, and elute the fusion protein using elution buffer.
  11. Collect the eluent and dialyze the protein sample against dialysis buffer overnight at 4C. > Optional: adding protease inhibitor cocktail tablets will help DNMT1 from proteolytic degradation.
  12. Take out sample from dialysis tube, spin down the sample and remove any precipitate
  13. Load the supernatant to the Heparin column, then apply the salt gradient with Buffer A and B. The target protein is expected to elute at 0.4-0.5 M NaCl.
  14. Collect the fractions and run the SDS Page to examine the protein (~100 kD). Pool together the protein fraction with significant purity
  15. Concentrate the fractions to about 1 ml using concentration spin columns (25MWCO)
  16. The protein is finally purified through a 16/60 Superdex 200 size exclusion column using Gel filtration buffer
  17. After the gel filtration, the protein will be examined by SDS page. The fractions with >90% purity will be pooled
  18. Concentration of sample and storage at -80C freezer.

Make competent cells already carrying two plasmid

  1. Do some stuff

Pick a colony without any plasmid from an agar plate

  1. Pick a single colony from the agar plate using a sterile pipette tip.
  2. Resuspend the colony in appropriate medium to make a culture or in sterile water.

Pick a colony carrying two plasmids from an agar plate

  1. Pick up a sterile tip with a pipette. Dip it into a single colony so it attaches to the tip without applying suction.
  2. Resuspend the colony in sterile medium or water.

Make a glycerol stock of bacteria carrying three plasmids

  1. Mix an aliquot of a LB culture with the same volume of storage buffer.
  2. Freeze at -80 °C.

CPEC

  1. Mix the DNA fragments at the desired ratio. Optionally add a small amount of DMSO.
  2. Add 2X Phusion Flash Master Mix
  3. Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.
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