Template:NYMU-2015notebook-protocol

Consistent tube labeling

Labeling tubes and plates in a consistent manner expedites the experimental process, and decreases the time taken to understand what others have done.

1.5ml microcentrifuge tubes

These 1.5ml microcentrifuge tubes are used to store extracted plasmids, cleaned up PCR or digested products, and frozen liquid culture.

Labeling:

  • On top:
    • Item name
    • Concentration (in ng/uL written in red)
    • "(PCR)" for PCR products; "(XP)","(SP)","(ES)","(EX)" for digestions
  • On the side:
    • Date (YYYY-MM-DD)
    • Person who made it
    • In red pen: the A260/280 and A260/230 ratios.

200uL microcentrifuge tubes

200uL microcentrifuge tubes (or PCR tubes) are used as temporary storage of various reactions.

Labeling:

  • Folding part: Write the day of the month.
  • On top: Write something that can identify what it is .

Agar plates

Agar plates are for transformations and obtaining single colonies.

Labeling:

  • On top (agar side):
    • Full item names (not just numbers!), date (YYYY-MM-DD), time of incubation start.
    • Additionally for transformation plates: Name of person who did the plating.
  • On side (agar side): type of antibiotic (A/Amp, C/Cm or K/Kan).

PCR

For any kind of PCR, make sure to list:

  • The things that are PCR'd, and their expected PCR lengths
  • The PCR mix protocol: write the 10uL first, then to multiply to get the new mix total version
  • the PCR time protocol
  • how the gel was loaded. Which wells was loaded with which items.
  • Which items were correct and are wrong after the gel run, in as much detail as possible (e.g. was the output the length of a self-ligated vector?)

Two-day efficient cloning cycle

We used an efficient two day cloning cycle split into a "Light" day and a "Heavy" day.

Approximate Light day:

  • 10:00am (~1hr): 3-in-1, put liquid and plate in fridge.
  • Lunch
  • After lunch (~1hr): Run Gel
  • 5:00pm: Incubate

Approximate Heavy day:

  • 10:00am (2-3hrs): Plasmid extraction + Digestion
  • Lunch (while digesting)
  • After lunch (2-3hrs): Gel Extraction/Purification + Ligation + Transformation
  • 5:00pm: Incubate plate

Light Day

The light day consists of Colony PCR and liquid culture of colonies transformed from a previous day.

  1. The 3-in-1

    2. The 3-in-1 protocol consists of a combination of three things:

    • Colony PCR
    • Liquid culture
    • 2nd time plate

    Preparation

    First, count the number of colonies you want to check.

    1. Make the Colony PCR mix (we use Thermo' DreamTaq) with the mix amount slightly modified:
      Item uL
      Template 1
      FP (VR) + RP (VF2) 2
      dNTP (2mM) 2
      10x DreamTaq buffer 5
      Taq Polymerase 0.2
      ddH20 39.8
      Total 50
    2. Aliquot 4 mL of LB medium in a centrifugal tube for each colony.
    3. Take out new plates from the fridge and divide them into smaller sections. This is the second-time plate. Make sure to write the date on it. Cross out in red pen any sections you know is wrong after the Colony PCR check

        Steps:

      1. Select a colony using a tip or toothpick.
      2. Dip it in a PCR tube and swirl it around.
      3. Use the same tip and dip it in the LB culture and swirl it around.
      4. Use the same tip and draw on the 2nd time plate.

        5. PCR run protocol

        Temperature Time
        94oC 60s
        94oC 15s 30-35 cycles
        55oC 20s
        72oC 1kb/min + 5-10s
        72oC 300s

  2. Making Gel for gel electrophoresis
    1. Make either 100ml or 200ml of gel at at time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)
    2. Measure out the relevant percentage in agarose (e.g. 2g of agarose for 100ml of 2% gel).
    3. Fill it up with 1xTAE buffer.
    4. Microwave on low, constantly taking it out every so often to swirl and mix it. Keep microwaving until clear. Make sure it is completely clear
    5. Cool to a temperature that is still hot but still can be held in your hand. If the gel gets too cold and starts to harden, start microwaving again until clear.
    6. Add 5uL of Safe-seeing dye for every 100ml of gel after cooled to the correct tempearature. Mix it well by swirling
    7. Pour it onto the molds quickly. Put a cover on it to block out the light.
    8. Wait at least 1/2hr until using it.
    9. Store in 4o and away from light.

  3. Gel electrophoresis
    1. Select a relevant % agarose gel based on your own experience (general guide: all <1000: 2%, all >2000: 1% guide)
    2. Load 5uL from each tube of Colony PCR, mix it with 1uL of 6x DNA Dye and put it in a well.
    3. Load 3uL of marker into a well.
    4. Run in the 13x13cm box at 90V or 100V and 400mA for the desired amount of time.
    5. View in the gel viewer machine.

  4. Liquid culture

    5. After confirming which colonies are correct from running the colony PCR on gel and deciding which ones to culture in liquid:

    1. Aliquot 6-15ml of LB in a 15ml centrifuge tube or 10ml of LB in a 50ml centrifuge tube for each colony.
    2. Add the 20uL of LB containing the correct colony from the 3-in-1 into the liquid.
    3. Add the relevant amount of antibiotics into the liquid. The volume of antibiotics added depends on the working and stock concentrations.
    4. Split the LB in the 15ml centrifuge tubes into multiple 15ml centrifuge tubes containing a maximum of 3ml each.
    5. Grow overnight at 37oC at 180rpm in the incubator.

Heavy day

The heavy day consists of extracting a previously grown plasmid, digesting it, and ligating digestions together, then transforming.

  1. Plasmid extraction

    2. We use GeneAid's plasmid extraction kit. We followed the protocol with the addition of warming the relevant amount of EB to 60-70oC before elution. Elute with 33uL.

  2. Digestion

    3. Thermo's fast-digest enzymes:

    Item XP from plasmid XP from PCR
    DNA 1ug or 10uL 10uL
    10x fast-digest (FD) buffer 2 (green) 2 (white)
    Thermo XbaI 0.5
    Thermo PstI 0.5
    ddH2O to 20uL 6
    Total 20

    Theoretically 10mins should be enough for this digestion. Over 1hr is better. NEB enzymes:

    Item SP EX ES
    DNA 1ug or 10uL
    10x Buffer 2 (Cutsmart) 2 (EcoRI)
    NEB Enzyme1 0.5
    NEB Enzyme2 0.5
    ddH2O to 20uL
    Total 20
  3. Digest for at least 1hr. Over 2hr is better. EcoRI doesn't have star activity when cut this way (even overnight)

    4. We run Insert digests (ES, XP) on a gel and then use GeneAid's gel extraction kit. We directly use GeneAid's PCR purification kit for vector digests (EX,SP). The modifications in the protocol include:

    • Warm EB to 60-70oC before elution.
    • Always use the Gel (sequencing) protocol for gel extraction.
  4. Ligation

    5. We use Thermo's and NEB's T4 Ligase:

    Item amount
    Vector 8.5uL, total of approximately 100ng of DNA.
    Insert
    ddH2O
    10x Ligase Buffer 1
    T4 Ligase 0.5
    Total 10

    Incubate at room temperature for 2hr then transform 1uL then put the remaining amount in a small bag and put in 4oC overnight in case the transformation fails and retransformation is required.

  5. Transformation

    6. We majorly use commercial E. coli DH5α competent cells.

    1. Add 2uL of plasmid or ligation mix to 20 uL of competent cells.
    2. Put mixture on ice for 30 minutes.
    3. Heat shock at 42 oC for 45 seconds
    4. Put the mixture back on ice for another 20 minutes
    5. Add 200 uL of LB broth to repair the cell wall; incubate at 37oC for 1.5 hr
    6. Plate it on a relevant antibiotic plate.
    7. Incubate plate at 37oC overnight.

Promoter testing

These are the protocols we used for promoter testing

General Gene Reporter Assay protocol

This is our general setup for gene reporting assays.

  1. Previous-night Preparation
    • Culture overnight (~16hrs) at 37C and shaking at 180rpm 2-3ml of single colonies of things to be measured.

      Preparation (can be done the previous night too):

      Determine the number of measurement points = number of items x number of replicates
    • Prepare that amount of 15ml round-bottom tubes and 1.5uL microcentrifuge tubes.
    • Add 2ml of LB + the relevant antibiotics in each round-bottom tube.
  2. Pre-start
    • Prewarm the LB+antibioics to the temperature to test at (e.g. 20mins prewarming in the incubator takes it up to 37C).
    • Get some ice buckets and pre-cool down the 1xPBS
  3. Culturing
    • Add any relevant chemicals to the prewarmed round-bottom tubes containing 2ml LB.
    • Dilute the overnight culture to an OD600 of about 0.0325
    • Incubate at the required conditions (usually 37C and 180rpm).
    • Repeat for every replicate.
  4. Measurement point retrieval

    5. For each culture tube:

    1. Take out the relevant culture and put it on ice
    2. Extract 1.3ml from the culture tube and put it in a 1.5uL microcentrifuge tube
    3. Centrifuge for 1min at 16.2g (13.2krpm on tabletop centrifuge)
    4. WASH: Tip out the supernatant and add 650uL of 1xPBS, resuspend, then centrifuge again.
    5. WASH again.
    6. Carefully remove and discard all the supernatant, resuspend in 1.3ml of 1xPBS.
    7. Store at 4C until measuring
  5. Measuring

    6. Requires black plates for measuring fluorescence and transparent plates for measuring OD600. For each measurement point, load 200uL each into 3 black wells and 3 transparent wells (i.e. 3 technical replicates each for fluorescence and OD600). Measure the fluorescence on the black plate with the settings depending on your machine. We use excitation/emission wavelength of 485/525 (100ms per measurement). Measure the OD600 on the transparent plate.

Transfection of plasmid DNA in plant cells

Production of tobacoo BY-2 protoplast

  1. 20 ml BY-2 suspension cellsa.
  2. Transfer cells to a sterile conical centrifuge tube, 3000 rpm, 10 mins.
  3. Remove the supernatant solution, and suspend cells by 10 - 20 ml protoplast digestion enzyme.
  4. Transfer the solution to 90×20 mm sterile plastic Petri dishes, incubate in the dark with gentle shaking (40 rpm) for 3-4 hours at room temperature.
  5. Filter the enzyme solution containing protoplast with a 40 μm nylon mesh.
  6. Place the filtered solution in a sterile 50 ml conical tube, centrifuge 250 ×g 5 mins.
  7. Gently remove the supernatant solution.
  8. Add 20 ml Protoplast Isolation Solution, gently suspend the protoplasts. Centrifuge the cells at 250 ×g 5 minutes.
  9. Resuspend the protoplast in 10 ml Protoplast Floating Solution. 250 ×g 10 mins. In this solution, the protoplasts will float to the top of the tube, while any non-protoplasted cells will pellet.
  10. Gently remove the floating protoplasts with a sterile dropper and transfer to a sterile conical tube.
  11. Add 10 ml Electroporation Solution. 250 ×g 5 mins. The protoplasts will pellet in this solution. Gently remove the supernatant.
    #
  • The BY-2 cell stage is at the beginning of linear phase; the cells were harvested after 3-d culture.
  • For one PEG reaction (cell density =1×105/ml) could be start from 20 ml BY-2 cells to isolate protoplasts.
  • Tips should be removed the top to avoid the stressful pipeting.

PEG transfection

  1. Add 10 ml cold W5 solution. 250 ×g 5 mins.
  2. Gently remove the supernatant and resuspend the protoplasts in W5 solution and adjust density to 1×106/ml. Keep protoplasts on ice for 30 minutes.
  3. Prepare the PEG solution.
  4. Centrifuge the protoplast suspension at 250 ×g for 5 mins. Remove the supernatant and add MMg solution and adjust density to 1×106/ml.
  5. Mix 10 μl DNA (10 μg) and 100 μl protoplasts.
  6. Add 110 μl PEG solution and mix well. Keep it at room temperature for 30 minutes.
  7. Add 1ml W5 solution and mix well. Centrifuge the protoplast suspension at 250 xg for 5 minutes.
  8. Remove the supernatant and add 1ml incubation solution. Place it in the dark for 16 hours at 26℃.

Solution preparing

  • Cell culture medium (BY-2 medium)(per liter)
    • 4.3 g MS salts
    • 0.1 g MS Vitamin mixture
    • 2 mg 2,4-dichloro-phenoxyacetic acid (2,4-D; an auxin)
    • 30 g sucrose
  • PEG solution (40%, w/v)
    • 0.4 g PEG 4000 (Fluca)
    • 0.6 ml 1 M mannitol
    • 100 μl 1 M CaCl2
  • W5 solution
    • 154 mM NaCl
    • 125 mM CaCl2
    • 5 mM KCl
    • 2 mM MES, pH 5.7
  • MMg solution
    • 0.6 M mannitol
    • 15 mM MgCl2
    • 4 mM MES, pH 5.7
  • Incubation solution
    • BY-2 medium
    • 0.4~0.6 M mannitol

Bacterial Conjugation

Shewanella (S.) oneidensis MR-1 Wild Type (WT) was conjugated with E. coli S17-1 strain having the plasmid of interest to infect and introduce plasmid into S. oneidensis MR-1 WT. In detail, pBBRI MCS-2 empty and engineered pBBR1 MCS:OmcA plasmids were transformed into E. coli SM strain prior the bacterial conjugation using standard biological techniques.


  1. An overnight culture of S. oneidensis MR-1 (MR-1 ) at 25°C and 150 rpm was prepared by selecting an individual colony from a plate of LB agar with the aid of a sterilized wire loop and inoculating it in 10 mL of LB.
  2. At the same time, an overnight culture of both transformed E. coli S17 strains was prepared also by selecting an individual colony from a plate of LB agar and inoculating it in 10 mL+kanamycin.
  3. Next day, harvest the cells by centrifugation (4000 rpm for 10 min) and wash the cell pellets twice with sterilised 1X PBS to remove antibiotics. Resuspend each cell pellet by 0.5 mL PBS and then mixed together.
  4. For the mating, 200 μ\- of the 1 mL cell suspension were spread onto LB agar plate (no antibiotics) and left at 25°C for 8 hours for the mating to happen.
  5. The cells were then scraped off the LB agar with a cell spreader and 1 ml 1X PBS and transferred to a clean eppendorf. Serial dilutions of 200 μ\- were plated onto LB agar with selection antibiotics (Ampicillin and Chloramphenicol) and incubated at 25°C.
  6. Successful colonies were restreaked in the same LB agar plates.

Extraction of Genomic DNA


Step 1. Cell harvest / Prelysis (For Gram-Negative bacteria)
Transfer bacterial culture (< 109 ) into a microcentrifuge tube. Centrifuge at full speed (13,000 rpm) for 1 min and discard the supernatant.

Step 2. Cell Lysis
(1) Add 200 μl of GB Buffer to the 1.5 ml microcentrifuge tube then vortex 5 seconds.
(2) Incubate at 60ºC for at least 10 minutes to ensure the sample lysate is clear.
During incubation, invert the tube every 3 minutes.
(3) RNA Degradation (If RNA-free gDNA is required, perform this optional step)
Following 60ºC incubation, add 5 μl of RNase A (10 mg/ml) to the clear lysate then mix by shaking vigorously.
(4)Incubate at room temperature for 5 minutes.

Step 3. DNA Binding
(5)Add 200 μl of absolute ethanol to the lysate then immediately mix by vortex for 10 seconds.
(6)Place a GD Column in a 2 ml Collection Tube.
(7)Transfer the mixture (including any precipitate) to the GD column.
(8) Centrifuge at 14-16,000 x g for 2 minutes.
(9) Discard the 2 ml Collection Tube then place the GD Column in a new 2 ml Collection Tube.
Step 4. Wash
(10)Add 400 μl of W1 Buffer to the GD Column then centrifuge at 14-16,000 x g for 30-60 seconds.
(11) Discard the flow-through then place the GD Column back in the 2 ml Collection Tube.
(12)Add 600 μl of Wash Buffer (make sure ethanol was added) to the GD Column.
(13) Centrifuge at 14-16,000 x g for 30-60 seconds then discard the flow-through.
(14)Place the GD Column back in the 2 ml Collection Tube.
(15) Centrifuge again for 3 minutes at 14-16,000 x g to dry the column matrix.

Step 5. DNA Elution
(16) Transfer the dried GD Column to a clean 1.5 ml microcentrifuge tube.
(17) Add 30-50μl of pre-heated Elution Buffer to the center of the column matrix.
(18)Let stand for at least 3 minutes to ensure the Elution Buffer or TE is completely absorbed.
(19) Centrifuge at 14-16,000 x g for 30 seconds to elute the purified DNA.