Team:Bilkent-UNAMBG/Project/protocols

COMPETENT CELL PREPARATION

 

  • Incubate the cells in 10 ml LB medium overnight at 37°C.
  • Dilute the overnight culture with 1:100 ratio.
  • Incubate for 2 hours 37°C until OD600 value is in between 0.2-0.6.
  • Split the culture into two ice-chilled falcon tubes and chill the samples on ice for 10 minutes.
  • Centrifuge the samples for 10 minutes at 4200 g at 4°C and remove the supernatant.
  • Resuspend the cells by pipetting with ice-chilled TSS buffer 1:10 ratio. NOTE: Perform the step on ice.
  • Add 100 μl of resuspended sample to ice-chilled 1.5 ml microcentrifuge tubes. Store at -80 °C. NOTE: Perform the step on ice.

 

 

TRANSFORMATION

 

  • Competent cells are put on ice until they are deforested. Add 100 ng of plasmid except the control group and keep the cells on ice for 30 minutes.
  • Put the cells on the heat block for 45 seconds at 42°C, then put them back on the ice for 2 minutes.
  • Add 250 μl LB medium.
  • Incubate the cells for 45 minutes in 37°C incubator.
  • Centrifuge the cells at 5800 g for 5 minutes.
  • Remove the supernatant (approximately 100 μl supernatant should be left in the tube).
  • Resuspend the cells by pipetting and spread them on agar plates.
  • Incubate the agar plates overnight at 37°C.

 

 

MINIPREP

(MN Plasmid DNA Purification kit and  Thermo Scientific GeneJET Plasmid Miniprep kits were used during these experiments)

(Solution volumes are stated for high-copy plasmids and low-copy plasmids respectively)

(Centrifuges at 12000-13000 g unless stated)

 

 

  • Centrifuge the overnight cell culture (10 mL with 1:1000 antibiotic) for 5 minutes at 8000 g and remove the supernatant.
  • Add 250/500 μl resuspension solution  on top of the cell pellet and vortex until the cell pellet is dissolved.
  • Transfer the resuspended cells to a 2 ml microcentrifuge tube.
  • Add 250/500 μl of the lysis solution and invert the tube for 4-6 time and wait for 5 minutes. NOTE: Do not harshly mix the solution but gently invert the tube and do not incubate the mixture more than 5 minutes so as to avoid genomic DNA contamination.
  • Add 300/600 μl neutralization solution  and mix the solution immediately by inverting the tube 6-8 times, the solution should become cloudy. NOTE: Do not harshly mix the solution but gently invert the tube.
  • Centrifuge for 10 minutes.
  • Transfer 700 μl of the supernatant to the column and centrifuge for 1 minute, remove the flow through. NOTE: Do not touch the white precipitate during the transfer of the supernatant. NOTE: If there is remaining supernatant in the micro-centrifuge tube, repeat the step 7.
  • Add 500 μl wash buffer 1 and centrifuge for 1 minute remove the flow through.
  • Add 700 μl wash buffer 2 and centrifuge for 1 minute remove the flow through.

 

NOTE: If you have only one wash buffer, use it twice (700 ul).

 

  • Centrifuge the empty column for 1 minute to remove left ethanol.
  • Put the top of the column to a 1.5 ml microcentrifuge tube.
  • Put the column+microcentrifuge tube on the pre-heated heat block for 3 minutes at 65°C to remove ethanol.
  • Add 20 μl 65°C pre-heated ddH2O on the middle of the column and wait for 3 minutes.
  • Centrifuge the column+micro-centrifuge tube for 4 minutes at 13000 g.
  • Perform nanodrop analysis with 1-2 μl of the sample.
  • Store at -20 °C.

 

 

RESTRICTION ENZYME DIGESTION

Reaction Buffer

5 μl

Restriction Enzyme

1 μl

DNA

300-400 ng for visualization*,

1000 ng for cloning

ddH2O

Complete the reaction volume to 50 μl with the addition of ddH2O.

Incubate the mixture at 37 °C for 2-3 hours.

*If the DNA amount is lower, a smaller reaction volume should be adjusted to use a lower amount of restriction enzyme.

 

LIGATION

T4 DNA Ligase Buffer

2 μl

T4 DNA Ligase

1 μl

Plasmid

50 ng

Insert DNA

1:X plasmid-insert DNA ratio, depends on their length.

ddH2O

Complete the reaction volume to  20ul with the addition of ddH2O.

 

 

  • Gently mix the reaction.
  • For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
  • For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.
  • Heat inactivate at 65°C for 10 minutes. This isn’t really necessary if you are going to perform chemical transformation.
  • Chill on ice and transform 5-10 μl of the reaction into 50 μl competent cells.

 

 

GEL EXTRACTION

(MN Plasmid NucleoSpin Gel kit was used during these experiments)

     (Centrifuges at 12000-13000 g unless stated)

 

  • Weigh the 2 ml microcentrifuge tube and tare the scale.
  • Add the cut agarose gel piece into the micro-centrifuge tube and weigh the added agarose gel piece.
  • Add NT1 solution on top of the agarose gel piece. (1:2 gel weight(g) to solution(ul)) Then, put the micro-centrifuge tube on the heat block for 10-20 minutes at 50°C until the agarose gel is fully dissolved.
  • Add 700 μl of the dissolved mixture to a column and centrifuge for 1 minute, remove the supernatant. If there is dissolved mixture left, repeat the step until all the dissolved mixture is passed through the column.
  • Add 700 μl NT3 buffer and centrifuge for 1 minute, remove the supernatant.
  • Repeat the previous step.
  • Centrifuge the empty column for 1 minute to remove left ethanol.
  • Put the top of the column to a 1.5 ml microcentrifuge tube.
  • Put the column+microcentrifuge tube on the pre-heated heat block for 3 minutes at 70°C to remove ethanol.
  • Add 15 μl 70°C pre-heated ddH2O on the middle of the column and wait for 3 minutes.
  • Centrifuge the column+micro-centrifuge tube for 4 minutes at 14000 g.
  • Perform nanodrop analysis with 1-2 μl of the sample.
  • Store at -20 °C.

 

 

PCR

PCR Master Mix (4X)

ddH2O

66 μl

Reaction buffer

20 μl

Reverse primer

5 μl

Forward primer

5 μl

dNTP mix

2 μl

Polymerase

1 μl



 

  • The master mix is prepared in a PCR tube.
  • 25 μl of the master mix is transferred into a new PCR tube as the control group.
  • Template DNA is added into the remaining master mix.
  • Template added master mix is aliquoted into three PCR tubes.
  • Protocol prepared according to the polymerase used.

 

 

AGAROSE GEL PREPARATION AND ELECTROPHORESIS

For 1% agarose gel,

 

  • Weigh 0.65 grams of agarose and mix with 65 ml TAE buffer inside an Erlenmeyer flask.
  • Heat the mixture inside the microwave oven until the agarose is melted (2-3 minutes).
  • After the mixture becomes clear, cool it with running water.
  • Add 0.65 μl SYBR Safe to the melted agarose mixture. (1:1 agarose g:SYBR safe ul)
  • Pour the agarose mixture on top of the prepared gel tank.
  • Remove the bubbles.
  • Wait for 15-20 minutes until the agarose gel is solidified.
  • Add 6X purple loading dye to the samples and pipette gently, avoid bubble formation.
  • Load 5 μl 2log ladder into one of the wells and load the samples.
  • Connect the plugs and run the gel for 35 mins at 140V.

 

 

GIBSON REACTION

Gibson mix

7.5 μl

Plasmid

50 ng

DNA fragment

1:X plasmid-insert ratio, depends on their length

After the reaction mixture is prepared the, it is incubated for 1 hour at 50°C inside the PCR machine.



AGAR PLATE PREPARATION

(Work in a sterile environment near the Bunsen Burner flame.)

 

  • Put the LB agar medium on the heater at 150-200 °C and wait until it is dissolved.
  • Put the dissolved LB agar medium inside the pre-heated waterbed for 45 minutes at 50°C.
  • Add appropriate antibiotics with 1:1000 ratio.
  • Pour the LB agar medium on the agar plates, approximately 20 ml for each agar plate.
  • Wait until LB agar medium is solidified.
  • Plates can be used immediately or be stored at 4°C after they cool down.

 

 

LB MEDIUM AND LB AGAR MEDIUM INGREDIENTS

For 1 lt LB medium,

 

  • 10 g peptone/tryptone
  • 5 g yeast extract
  • 5 g NaCl

 

NOTE: Add 15 g agar for LB agar medium.

 

SDS GEL ELECTROPHORESIS

  1. Preparation of SDS gel

For a 5 ml stacking gel:

      1. H20 2.975 ml
      2. 0.5 M Tris-HCl, pH:6.8 1.25 ml
      3. 10%(w/v) SDS 0.05 ml
      4. Acrylamide/Bis-acrylamide (30%/0.8%) 0.67 ml
      5. 10% (w/v) ammonium persulfate (APS) 0.05 ml
      6. TEMED 0.005 ml

Note: APS and TEMED must be added right before each use.

For a 10 ml separating gel:

 

  1. We load 20 ul of the samples mixed with loading dye and run at 120 V.
  2. After the run we place the gel in a container with Coomassie Blue dye for staining and microwave for 15-40 seconds. The gel should be checked in between to be safe.
  3. The container is put on shaker for 10 minutes the washed under water.
  4. We then place the gel into another container with Destaining Buffer and it can be put on shaker overnight for the destaining. The bands are usually clearly visible the next morning.  

 

WESTERN BLOT

  1. Before setting up the cassettes we place Whatman filter papers into Transfer Buffer, and the nitrocellulose membrane into methanol first for a few minutes then into Transfer Buffer as well.
  2. One of the Whatman papers is placed in the cassette and flattened with the rolling apparatus gently so there are no bubbles trapped.
  3. The membrane is placed on to the first paper and the gel is placed onto the membrane. The second layer of Whatman paper goes onto the gel. After placing each layer we roll over them with the apparatus again gently to smooth out the bubbles.
  4. We place this cassette inside the tank and it is set to Turbo -> Mini Gel -> Run for a 7-minute transfer.
  5. While the transfer is occurring we prepare the Blocking Buffer with 5% dry milk in TBS-T. This solution can be used up to a few times so we do not discard it, keep it at -20.
  6. When the transfer is over we take the membrane out with tweezers gently and the top corner is marked to know which side is up.
  7. The membrane is placed inside the milk solution and incubated inside the shaker for 1-2 hours at RT.
  8. Then we place the membrane carefully into the 1st Antibody solution (1:10000 in 5% dry milk solution in TBS-T) and keep it rocking on the shaker for another 1 hour at RT or O/N at +4 (cold room).
  9. Remove primary Ab solution and keep it at -20 (up to 5 usages) then we put enough TBS-T to cover it, leaving it rocking for another 5 minutes. Afterwards the membrane is washed twice, first for 5 then for 10 minutes.
  10. Then we expose the membrane to the 2nd Antibody (1:10000 in 5% dry milk solution in TBS-T) for 1 hour at RT, rotating.
  11. Remove secondary Ab solution and keep it at -20 (up to 5 usages) then we put enough TBS-T to cover it, leaving it rocking for another 5 minutes. Afterwards the membrane is washed twice, first for 5 then for 10 minutes.
  12. Then we take the image of the membrane.
  13. DO IT IN DARK!!! Take 500ul A and B from ECL solution substrates and mix. Apply the solution on membrane and wait for 1 min. Remove the substrate and take the image.

 

COLUMN REGENERATION

  1. Add 700 μl of 1 M HCl to previously used columns, close their lids and wait for 3 days.
  2. Remove the HCl inside the column and the collection tube.
  3. Centrifuge all columns for 1 minute at max speed.
  4. Add 700 μl ddH2O, centrifuge for 1 minute at max speed.
  5. Repeat step 4.
  6. Add 700 μl QBT, centrifuge for 1 minute at max speed.
  7. Repeat step 6.
  8. Repeat steps 4 and 5.
  9. Centrifuge empty columns for 3 minutes at max speed.
  10. Open the lids of the columns and let them dry.