Difference between revisions of "Team:NTNU Trondheim/Experiments"

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             </div>
 
             </div>
−
             <p>Note title</p></div>
+
             <p>Separation, isolation and transformation of linear DNA (pgRNA with anti-luxS)</p></div>
−
             <div class="collapsible-body"><span></span></div>
+
             <div class="collapsible-body"><span>
 +
<b>Goal:</b>
 +
<br>
 +
<ol>
 +
<li> Separate the PCR product from circular DNA</li>
 +
 
 +
<li>Isolate the linear DNA (pgRNA with anti-luxS from the gel</li>
 +
 
 +
<li>Transform the linear DNA into competent cells</li>
 +
 
 +
<li>InterLab study - transformation of plasmids to competent cells</li>
 +
</ol>
 +
<br>
 +
<b>Procedure:</b>
 +
<br>
 +
<u>Separation of linear DNA from circular DNA:</u>
 +
<br>
 +
The leaner DNA obtained from PCR (from day 8 – 29. June. 2018), was separated from the circular DNA (original plasmids) after running the sample (25μL) mixed with loading dye (5μl) on gel electrophoresis (gelGreen) simultaneously with a ladder (1μL gene ruler + 1μL loading dye + 4μL dH2O) for 50min at 90V. The sample and the ladder were prepared after the same protocol from day 3 – 22. June. 2018.
 +
 
 +
<br>
 +
<br>
 +
 
 +
<u>Gel digestion and DNA isolation:</u>
 +
<br>
 +
The agarose was digested and the DNA was isolated from the sample after the following protocol:
 +
<ol>
 +
<li>Add 3 volumes of ADB buffer to each volume of agarose excised from gel.</li>
 +
<li>Incubate the sample for 5-10min at 50℃.</li>
 +
<li>Transfer the melted sample to column with collection tube. Centrifuge for 60s at 16000g. Discard the flow-through (repeat this step until all solution of the sample has been transferred to the column).</li>
 +
<li>Add DNA wash buffer (200μL). Centrifuge for 30s at 16000g (the DNA in the column was washed twice by repeating this step) </li>
 +
<li>Discard the flow though</li>
 +
<li>Add d H­2O (20μL) directly to the column matrix. Centrifuge for 60s at 16000g. (The collected flow- through was transferred back to the column matrix, and centrifuged and collected in Eppendorf tube).</li>
 +
</ol>
 +
 
 +
<u>Determine the DNA concentration and purity:</u>
 +
<br>
 +
 
 +
The concentration and the purity of the isolated DNA was determined by using Nanodrop.
 +
 
 +
<br>
 +
<br>
 +
 
 +
<u>Transformation of PCR product to competent cells:</u>
 +
The PCR product (5μL) was transformed to competent cells after following the transformation protocol from day 4 – 25. June. 2018).
 +
<br>
 +
<br>
 +
 +
 
 +
<u>InterLab – transformation of plasmids to competent cells:</u>
 +
<br>
 +
The plasmids listed on the protocol, was transformed to competent cells (DH5α) after the following protocol:
 +
<br>
 +
<ol>
 +
<li>Thaw the competent cells on ice (10min), and pre-chill the eppendorf tubes.</li>
 +
<li>Resuspend the DNA in the wells (provided by iGEM HQ) with d H2O (10μL).</li>
 +
<li>Add competent cells (50μL) to the eppendorf tubes.</li>
 +
<li>Add resuspended DNA (1μL) to the Eppendorf tubes.</li>
 +
<li>Incubate on ice for 25min</li>
 +
<li>Heat shock the cells for 45s at 42℃ in water bath</li>
 +
<li>Incubate on ice for 5min.</li>
 +
<li>Add LB-medium (950μL) to the tubes</li>
 +
<li>Incubate for 1h at 37℃ with shacker</li>
 +
<li>Pipette each sample (100μL) to agar plate with CM</li>
 +
<li>Centrifuge the samples for 3min at 6800g, discard supernatant (800μL) and resuspend the pellet with the remaining supernatant</li>
 +
<li>Pipette each sample (100μL) to agar plate with CM</li>
 +
<li>Incubate overnight at 37℃.</li>
 +
</ol>
 +
<br>
 +
<br>
 +
<b>Results:</b>
 +
<br>
 +
 
 +
</span></div>
 
           </li>
 
           </li>
 
           <li>
 
           <li>
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             <p>InterLabs Study</p></div>
 
             <p>InterLabs Study</p></div>
−
             <div class="collapsible-body"><span> Calibration 1 and 2 for the InterLabs Study.</span></div>
+
             <div class="collapsible-body"><span> Calibration 1 and 2 for the InterLabs Study.
 +
 
 +
<b>Goal:</b>
 +
<br>
 +
 
 +
<b>Procedure:</b>
 +
<br>
 +
<b>Results:</b>
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<br></span></div>
 
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             <p>Note title</p></div>
 
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             <div class="collapsible-body"><span>Lorem ipsum dolor sit amet.</span></div>
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             <div class="collapsible-body"><span><b>Goal:</b>
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 +
<b>Procedure:</b>
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<br>
 +
<b>Results:</b>
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<br>
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<b>Procedure:</b>
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<br>
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<b>Results:</b>
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<br>
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<b>Procedure:</b>
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<b>Results:</b>
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Revision as of 08:45, 12 July 2018

Lab Journal


  • Week
    25

    Getting started

    • Jun
      18

      No lab

      No lab.
    • Jun
      19

      No lab

      No lab.
    • Jun
      20

      Our first day in the laboratory!

      Goal: Incubate the Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 on agar plates with antibiotics.

      Preparation of LA- medium
      LA- medium (1.5%) were made after the following recipe, and autoclaved for 20min at 121°C.
      • 1L Distilled water
      • 5g Yeast extract
      • 10g Tryptone
      • 5g NaCl
      • 15g Agar

      Agar plates with antibiotics
      Two different agar plates were made – one type contains ampicillin (AMP) while the other type contains chloramphenicol (CM), and were prepared after the following recipe:

      LA- medium with AMP:
      • 0.5L LA-medium
      • 0.5mL AMP (50mg/mL)
      LA-medium with CM:
      • 0.5L LA-medium
      • 0.5mL CM (35mg/mL)

      The LA-media with antibiotic were poured on petri dishes.

      Incubation of bacteria
      Two Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 plasmids respectively, were ordered from Addgene. E.coli which carry the pgRNA plasmid were plated on agar plates with AMP, while E.coli which carry the pdCas9 plasmid were plated on two agar plates with CM, and incubated at 37℃.

      Results:
      The E.coli with pgRNA and pdCas9 formed colonies on the agar plates with AMP and CM
    • Jun
      21

      Inoculation of E.coli

      Goal: Inoculate a colony from each agar plate which were prepared from the previous day, in LB- medium with antibiotics.

      Procedure
      One colony of pgRNA- and pdCas9- bacteria grown overnight on agar plates were picked and inoculated in LB-media (25mL) with AMP (25μL) and CM (25μL), respectively. The cell cultures were incubated at 37℃ in a shaking incubator at 204rpm.
    • Jun
      22

      Isolation and verification of pgRNA and pdCas9

      Goal: Isolate and verify that the E.coli carry pgRNA and pdCas9.

      Plasmid isolation
      pgRNA and pdCas9 were isolated from the incubated bacteria prepared yesterday (June 21) by following the protocol of ZR Plasmid Miniprep Kit:
      1. 1mL of each cell culture (which either carry pgRNA or pdCas9) were transferred to eppendorf tube and centrifugated for 20s at 16000g
      2. The supernatant was discarded, while the pellet was resuspended in buffer
      3. Lysing buffer was added to the sample and carefully mixed in 2min
      4. Neutralizing buffer was added to the sample, and mixed until the colour of the solution became yellow
      5. The solution was centrifugated for 2min at 16000g
      6. The supernatant was collected and transferred to column in a collection tube, and centrifugated for 30s at 16000g
      7. The supernatant in the collection tube was discarded
      8. Endo-wash buffer was added to the column and centrifugated for 1min at 16000g
      9. Plasmid- wash buffer was added to the column and centrifugated for 1 min at 16000g
      10. The solution in the collection tube was discarded, while the column with plasmids was transferred to a new collection tube
      11. The plasmids in the column were eluted by adding 30μL distilled water, and centrifugated for 30s in 16000g

      Determine plasmid concentration and purity
      The concentration of plasmids and the purity of each sample were determined by using Nanodrop.

      Restriction digest and plasmid verification
      The presence of pgRNA and pdCas9 in the cell cultures were verified after following the restriction digest protocol and separation of the DNA fragments by gel electrophoresis.
  • Week
    26

    Note title

    • Jun
      25

      Transformation of pgRNA and pdCas9 into competent cells

      Goal: Transform pgRNA and pdCas9 whinto competent cells (E.coli K-12 DH5α).

      Procedure:
      pgRNA and pdCas9 which were isolated from day 3 (22. June. 2018) were transformed into E.coli K-12 DH5α cells after the following transformation protocol:
      1. Thaw competent cells on ice for 10min.
      2. Use a pipette and transfer 2μL of mini-prepped plasmids into DH5α samples. Pipette up and down while stirring the pipette tip gently for 5-10s.
      3. Incubate the cells on ice for 20min.
      4. Heat shock the cells for 45s at 42℃. Immediately put the samples on ice for 2min.
      5. Add 900μL LB medium to the cells. Incubate at 37℃, 200RPM for 1h.
      6. Plate out 100μL of the transformation medium into agar plates with selection agent (in our case, we chose chloramphenicol).
      7. Incubate the cells at 37℃.

      Procedure:
      Sett inn bilder.
    • Jun
      26

      Transfer successful transformed cells to LB-medium and preparation of standard curves for interLab study

      Goal:
      Inoculate successful transformed competent cells to LB-medium, and prepare the standard curves for interLab study.

      Procedure:
      Inoculate colony from agar plates to LB-medium:
      A colony from each agar plate were selected and inoculated to LB- medium with antibiotics. Competent cells with pgRNA were transferred to LB- medium with AMP, while competent cells with pdCas9 were inoculated into LB- medium with CM. The cell cultures were incubated at 37℃ with shaker.

      Preperation of 1xPBS splution:
      1xPBS solution for the interLab study was made after the following recipe:
      • 1L Distilled water
      • 8g NaCl
      • 0.2g KCl
      • 1.42g Na2HPO4
      • 0.24g KH2PO4

      Prepareation of agar plates, LA- and LB-medium:
      LA- and LB- medium was made after the protocols from day 1 (20. June. 2018).
      Agar plates with CM were prepared after the protocol from day 1(20. June. 2018).

      Preparation of standard curves for interLab study:
      Calibration 1: OD600 reference point – LUDOX protocol
      A conversion factor to transform absorbance data from plate reader into OD600 was obtained in spectrometer by measuring 4 replicates of LUDOX CL-X and dd H2O. The data were imported into Excel sheet.
      Calibration 2: Particle standard curve – Microsphere protocol
      Solution of silica beads provided in kit from iGEM HQ, was vortexed before dilution with water (96μL Silica beads in 904μL dd H2O). A serial dilution of microspheres was obtained by adding 100μL dd H2O into 4x11 wells (E2, F2, G2, H2…E12, F12, G12, H12) on a 96 well plate. Microsphere stock solution (200μL) was pipetted into E1 and 100μL of the solution was transferred to E2 and mixed well before transferring 100μL to E3. The procedure repeated until 100μL solution was pipetted into E11, and 100μL was transferred to liquid waste. The dilution series was repeated for row F, G and H. The absorbance of the samples was measured by a plate reader with shaker at 24℃. The data were exported to Excel sheet.

      Results:
      Sett inn bilde her.
    • Jun
      27

      Isolation of pgRNA and pdCas9 from copentent cells (DH5alpha) and preparation of stock solution for calibration 3 for interLab study

      Goal:
      Isolate plasmids from competent cells (DH5α), and prepare the fluorecin stock solution for interLab study.

      Procedure:
      Dilution of cell cultures:
      Each cell cultures (successful transformed competent cells from day 5 – 26.06.2018) (2.5mL) were transferred into fresh LB-medium (22.5mL) with CM (22.5μL). The new cultures were incubated for about 2 hours at 37℃ with shaker.

      Measurement of the cell density:
      The cell density of the new cultures was measured at OD600, after calibration with water (blank sample).

      Plasmid isolation and determination of concentration:
      The plasmids were isolated from the competent cell after the protocol of ZR plasmid miniprep kit (see lab journal from day 3 - 22. June. 2018).
      The concentration of the plasmids was determined by using Nanodrop.

      Restriction digest:
      The plasmids from each culture (5μL) were digested by mixing with 10xbuffer (2μL), distilled water (12.5μL) and restriction enzyme BspHI (0.5μL). The solution was centrifuged down before the samples were incubated over night at 37℃.

      Results:
    • Jun
      28

      Plasmid verification and storing the cell cultures

      Goal:
      Verify that the successfully transformed DH5α cells carry pgRNA or pdCas9, and store the cell cultures for future use.

      Procedure:
      Plasmid verification:
      The digested plasmid samples from yesterday’s incubation (lab 6 – 27. June.2018) were separated by gel electrophoresis (see the protocols from lab 3 – 22. June. 2018).

      Bacteria glycerol stocks:
      The cell cultures made from 27. June were stored in glycerol stock after following protocol:
      1. Add cell culture (500μL) to glycerol stock (500μL, 50%) in cryo tubes.
      2. Freeze the samples at -80℃.

      The glycerol stock was prepared after following recipe:
      • 25mL Glycerol (99.56%)
      • 25mL Distilled water

      Results:
      Plasmid verification:
      L, pR1, pR2, pC1 and pC2 are the DNA-fragments from ladder solution, the two samples of pgRNA and pdCas9, respectively. From Figure 1 the two fragments with 1.5kb and 1.0kb were found from the fragments of pgRNA after digestion by BspHI. The fragment with 100bp from the digested pgRNA is not visible on the gel, probably due to masking of the dye colour at the bottom of the gel. Two bands from the digested pdCas9 with 2,7kb and 4kb were also detected. By comparing the results og gel elecrophoresis obtained from today's experiment with the results from day 3 (22. June. 2018), one can clearly see that the bands with the expected length from successful transformed competent cells, are much clearer. Hence, this also indicate the samples from the successful transformed DH5α cells are purer, which is also true by comparing the results from Nanodrop (see day 6 - 27. June. 2018, and day 3 - 22. June. 2018).
    • Jun
      29

      Insertion of ant-luxS via PCR and fluorescence standard curve for interLab study

      Goal:
      Insert anti-luxS in pgRNA plasmid, and generate fluorescence standard curve for the interLab study.

      Procedure:
      Insertion of anti-luxS gene and amplification of pgRNA:
      Reverse anti-luxS primer (26.2nmol) and forward anti-luxS primer (30nmol) was suspended with 261μL and 299μL water, respectively, to obtain 100μM solutions. Each primer (10μL) were diluted with water (990μL) to obtain a final concentration of 1μM.

      The 20bp of anti-luxS gene was inserted into pgRNA (2) via PCR after the following protocol:
      • 12.5μL Takara high five pre-mix (do not contain primers or templates)
      • 7.5pmol Primer (forward (FW) and reverse (RV) anti-luxS primers)
      • >100ng Template (we use >50ng)

      1. Forward anti-luxS primer (6μL) and reverse anti-luxS primer (6μL) were added to a PCR tube
      2. pgRNA (2) (0.5μL, 44.2ng/μL) was added to the PCR tube
      3. Takara pre-mix (2x6.5μL) was added to the solution
      4. The sample was amplified by PCR, and stored in freezer

      InterLab – Calibration 3
      A fluorescence standard curve was generated from the data obtained after measuring the fluorescence of the serial dilution of fluorescein.

      The fluorescence was measured with the following settings:

      Temperature: 24℃

      Gain (manual): 56

      Wave length [nm] Band width [nm]
      Excitation 494 4
      Emission 525 20

      Results:
      InterLab – Calibration 3:
      Figure 1 shows the florescence standard curve in the concentration interval [0, 2.5μM], with a R2 value of 0,9956. The model of the fluorescence standard curve is:
  • Week
    27

    Note title

    • Jul
      2

      Separation, isolation and transformation of linear DNA (pgRNA with anti-luxS)

      Goal:
      1. Separate the PCR product from circular DNA
      2. Isolate the linear DNA (pgRNA with anti-luxS from the gel
      3. Transform the linear DNA into competent cells
      4. InterLab study - transformation of plasmids to competent cells

      Procedure:
      Separation of linear DNA from circular DNA:
      The leaner DNA obtained from PCR (from day 8 – 29. June. 2018), was separated from the circular DNA (original plasmids) after running the sample (25μL) mixed with loading dye (5μl) on gel electrophoresis (gelGreen) simultaneously with a ladder (1μL gene ruler + 1μL loading dye + 4μL dH2O) for 50min at 90V. The sample and the ladder were prepared after the same protocol from day 3 – 22. June. 2018.

      Gel digestion and DNA isolation:
      The agarose was digested and the DNA was isolated from the sample after the following protocol:
      1. Add 3 volumes of ADB buffer to each volume of agarose excised from gel.
      2. Incubate the sample for 5-10min at 50℃.
      3. Transfer the melted sample to column with collection tube. Centrifuge for 60s at 16000g. Discard the flow-through (repeat this step until all solution of the sample has been transferred to the column).
      4. Add DNA wash buffer (200μL). Centrifuge for 30s at 16000g (the DNA in the column was washed twice by repeating this step)
      5. Discard the flow though
      6. Add d H­2O (20μL) directly to the column matrix. Centrifuge for 60s at 16000g. (The collected flow- through was transferred back to the column matrix, and centrifuged and collected in Eppendorf tube).
      Determine the DNA concentration and purity:
      The concentration and the purity of the isolated DNA was determined by using Nanodrop.

      Transformation of PCR product to competent cells: The PCR product (5μL) was transformed to competent cells after following the transformation protocol from day 4 – 25. June. 2018).

      InterLab – transformation of plasmids to competent cells:
      The plasmids listed on the protocol, was transformed to competent cells (DH5α) after the following protocol:
      1. Thaw the competent cells on ice (10min), and pre-chill the eppendorf tubes.
      2. Resuspend the DNA in the wells (provided by iGEM HQ) with d H2O (10μL).
      3. Add competent cells (50μL) to the eppendorf tubes.
      4. Add resuspended DNA (1μL) to the Eppendorf tubes.
      5. Incubate on ice for 25min
      6. Heat shock the cells for 45s at 42℃ in water bath
      7. Incubate on ice for 5min.
      8. Add LB-medium (950μL) to the tubes
      9. Incubate for 1h at 37℃ with shacker
      10. Pipette each sample (100μL) to agar plate with CM
      11. Centrifuge the samples for 3min at 6800g, discard supernatant (800μL) and resuspend the pellet with the remaining supernatant
      12. Pipette each sample (100μL) to agar plate with CM
      13. Incubate overnight at 37℃.


      Results:
    • Jul
      3

      InterLabs Study

      Calibration 1 and 2 for the InterLabs Study. Goal:
      Procedure:
      Results:
    • Jul
      4

      Note title

      Goal:
      Procedure:
      Results:
    • Jul
      5

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      Goal:
      Procedure:
      Results:
    • Jul
      6

      Note title

      Goal:
      Procedure:
      Results:
  • Week
    28

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      9

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      10

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    Note title

    • Aug
      13

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      14

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      15

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      16

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      17

      Note title

      Lorem ipsum dolor sit amet.
  • Week
    34

    Note title

    • Aug
      20

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      21

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      22

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      23

      Note title

      Lorem ipsum dolor sit amet.
    • Aug
      24

      Note title

      Lorem ipsum dolor sit amet.