Difference between revisions of "Team:NCKU Tainan/Protocol"
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<div class="collapse multi-collapse" id="COO"> | <div class="collapse multi-collapse" id="COO"> | ||
| + | |||
| + | <h3>Materials:</h3> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
| − | |||
pH meter, enzyme samples, magnetic stirrer, saturated CO2 solution, 20 mM Tris-HCl | pH meter, enzyme samples, magnetic stirrer, saturated CO2 solution, 20 mM Tris-HCl | ||
buffer (pH8.3), 20mL borosilicate glass vial | buffer (pH8.3), 20mL borosilicate glass vial | ||
| + | </p> | ||
| − | + | <h3>Method:</h3> | |
| − | + | <p class="pcontent"> | |
Saturated CO2 solution preparation</br></br> | Saturated CO2 solution preparation</br></br> | ||
Dissolve gaseous CO2 into deionized water (on ice) until it is saturated. (At least | Dissolve gaseous CO2 into deionized water (on ice) until it is saturated. (At least | ||
30 min)</br></br> | 30 min)</br></br> | ||
| − | 20 mM Tris HCl buffer (pH8.3) preparation</br></br> | + | |
| + | 20 mM Tris HCl buffer (pH8.3) preparation</br></br></p> | ||
| + | <ol> | ||
<li>Dissolve 121.14 g Tris in 800 ml deionized water.</li></br> | <li>Dissolve 121.14 g Tris in 800 ml deionized water.</li></br> | ||
<li>Add a pH meter into the solution to observe the pH.</li></br> | <li>Add a pH meter into the solution to observe the pH.</li></br> | ||
| Line 665: | Line 669: | ||
<li>Calculate CA activity using a Wilbur–Anderson unit (WAU) per milliliter of | <li>Calculate CA activity using a Wilbur–Anderson unit (WAU) per milliliter of | ||
sample.</li></br> | sample.</li></br> | ||
| + | </ol> | ||
| − | |||
</div> | </div> | ||
Revision as of 12:56, 29 September 2018
Protocol
- Gently mix the following reaction by pipetting and centrifuge briefly.
| 20 μl system | 50 μl system | |
|---|---|---|
| Template | 12～20 ng | 30～50 ng |
| Forward primer | 1.0 μl | 2.5 μl |
| Reverse primer | 1.0 μl | 2.5 μl |
| dNTP | 1.6 μl | 4.0 μl |
| 10x Buffer | 2.0 μl | 5.0 μl |
Program of KOD DNA polymerase
| Temperature | Time | |
|---|---|---|
| 94 ℃ | 3 min. | 25～30 cycles |
| 94 ℃(Denaturation) | 40 sec | |
| 57.5 ℃(Annealing) | 30 sec | |
| 72 ℃(Extension) | Depend on sequence size(2 kbp/min. for Taq) | |
| 72 ℃ | 5 min. | |
| 4 ℃ | ∞ |
- Confirm the size of the digested product by gel electrophoresis.
- Gel purification of the target size.
- Digestion (vector)
| Plasmid | 200 ng | 1000 ng |
|---|---|---|
| EcoRI / SpeI | 0.2 μl | 1 μl |
| XbaI / PstI | 0.2 μl | 1 μl |
| CutSmart Buffer | 2 μl | 5 μl |
| ddH2O | Up to 20 μl | Up to 50 μl |
| Digestion at 37℃ for 2.5hr. |
- Digestion (insert)
| Plasmid | 200 ng | 1000 ng |
|---|---|---|
| EcoRI / XbaI | 0.2 μl | 1 μl |
| SpeI / PstI | 0.2 μl | 1 μl |
| CutSmart Buffer | 2 μl | 5 μl |
| ddH2O | Up to 20 μl | Up to 50 μl |
| Digestion at 37℃ for 2.5hr. |
- Confirm the size of the digested product by gel electrophoresis.
- Gel purification of the target size.
- Ligation
| Vector (2 kbp) | molar ratio = 1:3(can up to 1:10 depending on the DNA sizes) |
| Insert (1.5 kbp) | |
| Quick Ligase Reaction Buffer (2X)* | 10 μl |
| Quick Ligase | 1 μl |
| ddH2O | Up to 20 μl |
- Transform the product by heat shock.
Gel Dissociation
- Gel Extraction
- Excised the DNA fragment from the agarose gel.
- Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube.
- Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex.
- Incubate at 55~60℃ for 10 minutes (or until the gel slice has completely dissolved).
- During the incubation, mixed by vortexing the tube every 2~3 minutes.
- Cooled the dissolved sample mixture to the room temperature.
DNA Binding
- Placed a PG Column in a Collection Tube. Apply the supernatant to the PG Column by decanting or pipetting.
- Centrifuged at 16,000 xg for 30 seconds.
- Discarded the flow-through and place the PG Column back into the same collection tube.
Wash
- Added 400 μl of the Buffer W1 into the PG Column.
- Centrifuged at 16,000 xg for 30 seconds.
- Discarded the flow-through and place the PG Column back into the same collection tube.
- Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.
- Centrifuged at 16,000 xg for 30 seconds.
- Discarded the flow-through and place the PG Column back into the same collection tube.
- Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer W2.
Elution
- To elute the DNA, placed the PG Column in a clean 1.5 ml microcentrifuge tube.
- Added 50 μl of the H2O (pH is between 7.0 and 8.5) to the center of each PG Column, let it stand for at least 2 minutes, and centrifuge at 16,000 xg for 2 min.
For E. coli DH5α,BL21(DE3) and W3100(DE3) competent cell
- Streak out wild type E. coli on a plate (LB plate without antibiotics) overnight and pick one colony into 3 ml of media (LB) and grow overnight.
- Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at 37 ℃.
- When the OD600 nm up to 0.35, put the cells on ice immediately.
- Spin the cells at 4℃for 10 minutes at 4000 rpm.
- Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer 1(TFB1).
- Leave nicely suspended bugs on ice for 10 minutes.
- Spin the cells at 4℃ for 10 min. at 4000 rpm.
- Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).
- Leave on immediately on ice for 30 minutes.
- Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with liquid nitrogen.
- Store the frozen cells in the -80℃ freezer.
- Transfer 1.4 ml of well-grown bacterial culture to a centrifuge tube.
- Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and discard the supernatant completely.
- Add 200 µl of FAPD1 Buffer (RNaseA added) to the cell pellet and resuspend the cells completely by pipetting.
- Make sure that RNaseA has been added into FAPD1 Buffer when first use.
- No cell pellet should be visible after resuspension of the cells.
- Add 200 µl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times. Incubate the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells.
- Do not vortex, vortex may shear genomic DNA. If necessary, continue inverting the tube until the lysate become clear.
- Make sure the tube transfer to clarify from turbid.
- Do not proceed with the incubation over 5 minutes.
- Add 300 µl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to neutralize the lysate.
- Invert immediately after adding FAPD3 Buffer will avoid asymmetric precipitation.
- Centrifuge at 16,000 xg for 3 minutess. to clarify the lysate. During centrifugation, place a FAPD Column in a Collection Tube.
- Transfer the supernatant carefully to the FAPD Column and centrifuge at 16,000 xg for 1 minute. Discard the flow-through and place the column back to the Collection Tube.
- Do not transfer any white pellet into the column.
- Add 400 µl of W1 Buffer to the FAPD Column and centrifuge at 16,000 xg for 1 minute. Discard the flow-through and place the column back to the Collection Tube.
- Add 600 µl of Wash Buffer to the FAPD Column and centrifuge at 16,000 xg for 1 minute. Discard the flow-through and place the column back to the Collection Tube.
- Make sure that ethanol (96 ~ 100 %) has been added into Wash Buffer when first use.
- Centrifuge at 16,000 xg for an additional 3 minutes to dry the FAPD Column.
- Important step! The residual liquid should be removed thoroughly on this step.
- Place the FAPD Column to a new 1.5 ml microcentrifuge tube.
- Add 30 µl of Elution Buffer or ddH2O to the membrane center of the FAPD Column. Stand the column for 3 minute.
- Important step! For effective elution, make sure that the elution solution is dispensed on the membrane center and is absorbed completely.
- Do not elute the DNA using less than suggested volume (50 µl). It will lower the final yield.
- Centrifuge at 16,000 xg for 3 minute to elute plasmid DNA and store the DNA at -20 ℃.
DNS solution preparation:
- Disolve 2.5g of 3,5-Dinitrosalicylic acid (DNS) to 150ml double distilled water.
- Heat to solution to 45 degree Celsius and add 4g of NaOH. Stir the solution until it is transparent.
- Add 75g of potassium sodium tartrate and add water to 250 ml.
- Keep the solution without light exposure. The solution can be used after 7 days.
Principle:
Under base solution, DNS will turn to brown color while reacting with reductive sugar in high temperature. In the specific temperature range, the color will have linear relationship with the reductive sugar concentration.
Calibration:
- Prepare glucose or xylose water solution to the following concentration: 0, 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2 g/L.
- Take 200 ul of sample, add 200 ul of DNS solution.
- Heat the sample at 100 degree Celsius for 10mins.
- Cool down the sample with ice to room temperature.
- Measure the optical density of the sample with 540nm light wavelength.
- Draw the graph of sugar concentration with respect to optical density.
Calibration results:
Measurement:
- Take 200 ul of sample, add 200 ul of DNS solution.
- Heat the sample at 100 degree Celsius for 10mins.
- Cool down the sample with ice to room temperature.
- Measure the optical density of the sample with 540nm light wavelength.
- Get the concentration of reductive sugar from the Calibration graph.
Materials:
pH meter, enzyme samples, magnetic stirrer, saturated CO2 solution, 20 mM Tris-HCl buffer (pH8.3), 20mL borosilicate glass vial
Method:
Saturated CO2 solution preparation Dissolve gaseous CO2 into deionized water (on ice) until it is saturated. (At least 30 min) 20 mM Tris HCl buffer (pH8.3) preparation
- Dissolve 121.14 g Tris in 800 ml deionized water.
- Add a pH meter into the solution to observe the pH.
- Slowly add concentrated hydrochloric acid (HCl) solution to reduce the pH to 8.3. Be careful not to add too much at a time, since the pH will change rapidly.
- Once the desired pH has been reached, top up the solution to 1 L using deionized water. Performing Blank Reaction
- Add 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer into a 20 mL borosilicate glass vial with further incubation at 0 °C with stirring.
- Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.
- Record the time course, T0 (in sec) of pH decrease from 8.3 to 6.3. The pH meter must be preset to 0°C and calibrated. Performing Test-Sample Reaction
- Mix 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer and 0.2 mL enzyme, transfer the mixture to a 20 mL borosilicate glass vial with further incubation at 0 °C with stirring.
- Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.
- Record the time course, T (in sec) of pH decrease from 8.3 to 6.3. The pH meter must be preset to 0°C and calibrated.
- Calculate CA activity using a Wilbur–Anderson unit (WAU) per milliliter of sample.
- Preculture the bacteria overnight by picking up a colony in 4ml LB with antibiotic.
- Culture the bacteria in 30 ml LB by adding 1/100 of bacteria , 1/2000 kanamycin, 1/4000 chloramphenicol to log phase( about 3 hr).
- Centrifuge them in 22℃ ,3200xg for 5 minutes , then refresh by the M9 medium with 4%xylose and 0.1%LB.
- Culture the bacteria for 24 hr in both O2 and 5%CO2 incubator , and test the O.D. value at every 6 hours.