Team:NCKU Tainan/Protocol

Protocol

PCR Plasmid Construction PCR Clean-Up & Gel Extraction Plasmid Extraction DNS reductive sugar measurement pH24 pH30 Carbonic Anhydrase Activity Assay Total Solution

PCR

1. 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.
94 ℃ (Denaturation)	40 sec	25～30 cycles
57.5 ℃ (Annealing)	30 sec
72 ℃ (Extension)	Depend on sequence size (2 kbp/min. for Taq)
72 ℃	5 min.
4 ℃	∞

2. Confirm the size of the digested product by gel electrophoresis.


3. Gel purification of the target size.

Plasmid Construction

1. 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.

2. 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.

3. Confirm the size of the digested product by gel electrophoresis.


4. Gel purification of the target size.


5. 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

6. Transform the product by heat shock.

PCR Clean-Up & Gel Extraction

Gel Dissociation

1. Gel Extraction
1. Excised the DNA fragment from the agarose gel.


2. Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube.


3. Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex.


4. Incubate at 55~60℃ for 10 minutes (or until the gel slice has completely dissolved).


5. During the incubation, mixed by vortexing the tube every 2~3 minutes.


6. Cooled the dissolved sample mixture to the room temperature.

DNA Binding

2. Placed a PG Column in a Collection Tube. Apply the supernatant to the PG Column by decanting or pipetting.


3. Centrifuged at 16,000 xg for 30 seconds.


4. Discarded the flow-through and place the PG Column back into the same collection tube.

Wash

5. Added 400 μl of the Buffer W1 into the PG Column.


6. Centrifuged at 16,000 xg for 30 seconds.


7. Discarded the flow-through and place the PG Column back into the same collection tube.


8. Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.


9. Centrifuged at 16,000 xg for 30 seconds.


10. Discarded the flow-through and place the PG Column back into the same collection tube.


11. Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer W2.

Elution

12. To elute the DNA, placed the PG Column in a clean 1.5 ml microcentrifuge tube.


13. 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.

Competent Cell Preparation

For E. coli DH5α,BL21(DE3) and W3100(DE3) competent cell

1. 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.


2. Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at 37 ℃.


3. When the OD600 nm up to 0.35, put the cells on ice immediately.


4. Spin the cells at 4℃for 10 minutes at 4000 rpm.


5. Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer 1(TFB1).


6. Leave nicely suspended bugs on ice for 10 minutes.


7. Spin the cells at 4℃ for 10 min. at 4000 rpm.


8. Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).


9. Leave on immediately on ice for 30 minutes.


10. Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with liquid nitrogen.


11. Store the frozen cells in the -80℃ freezer.

Plasmid Extraction

1. Transfer 1.4 ml of well-grown bacterial culture to a centrifuge tube.


2. Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and discard the supernatant completely.


3. 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.


4. 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.


5. 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.


6. Centrifuge at 16,000 xg for 3 minutess. to clarify the lysate. During centrifugation, place a FAPD Column in a Collection Tube.


7. 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.


8. 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.


9. 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.


10. 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.


11. Place the FAPD Column to a new 1.5 ml microcentrifuge tube.


12. 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.


13. Centrifuge at 16,000 xg for 3 minute to elute plasmid DNA and store the DNA at -20 ℃.

DNS Reductive Sugar Measurement

DNS solution preparation:

1. Disolve 2.5g of 3,5-Dinitrosalicylic acid (DNS) to 150ml double distilled water.


2. Heat to solution to 45 degree Celsius and add 4g of NaOH. Stir the solution until it is transparent.


3. Add 75g of potassium sodium tartrate and add water to 250 ml.


4. 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:

1. 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.


2. Take 200 ul of sample, add 200 ul of DNS solution.


3. Heat the sample at 100 degree Celsius for 10mins.


4. Cool down the sample with ice to room temperature.


5. Measure the optical density of the sample with 540nm light wavelength.


6. Draw the graph of sugar concentration with respect to optical density.

Calibration results:

Measurement:

1. Take 200 ul of sample, add 200 ul of DNS solution.


2. Heat the sample at 100 degree Celsius for 10mins.


3. Cool down the sample with ice to room temperature.


4. Measure the optical density of the sample with 540nm light wavelength.


5. Get the concentration of reductive sugar from the Calibration graph.

pH24

1. Preculture the bacteria o/n in LB, and prepared 8 different (pH4,4.25,4.5,4.75,5,5.5,6,7) pH value of M9 buffer.


2. Add 1/100 of the bacteria in 20 ml of different pH value of M9 buffer with 1/1000 chloramphenicol.


3. Culture the bacteria in the incubator in 37℃for 24 hr.


4. Measure the O.D. value (595 nm) and the fluorescence value (485-535nm ) at every 1 hr , within 24 hr.

pH30

1. Preculture the bacteria o/n in LB, and prepared 8 different (pH4,4.25,4.5,4.75,5,5.5,6,7) pH value of M9 buffer.


2. Culture the bacteria in 6 ml LB with 1/1000 chloramphenicol to log phase (about 1.5-2hr)


3. Divide 6 ml of bacteria into 8 eppendorfs, and centrifuged them at 1300 rpm for 1 mins in 37 ℃, then discard the supernatant completely.


4. Add 700 μl per different pH value of M9 buffer into 8 different eppendorfs, respectively . And resuspend the cells completely by pipetting.


5. Add 200 μl of bacteria in 96 well (This step need triple repetition.)


6. Measure the O.D.595 nm at the first and the last time point , and the fluorescence value (485-535nm ) at every 180 sec, within 30 mins.

Carbonic Anhydrase Activity Assay

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



1. Dissolve 121.14 g Tris in 800 ml deionized water.


2. Add a pH meter into the solution to observe the pH.


3. 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.


4. Once the desired pH has been reached, top up the solution to 1 L using deionized water.


• Performing Blank Reaction



5. 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.


6. Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.


7. 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



8. 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.


9. Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.


10. 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.


11. Calculate CA activity using a Wilbur–Anderson unit (WAU) per milliliter of sample.

Calculation:

Units/ml of enzyme= (T_{0 Average} - T_Average ) (df) / (T_Average )(E)

T = Time (in seconds) required for pH to change from 8.3 to 6.3 as per Unit Definition
df = dilution factor
E= volume (in milliliters) of enzyme used

Total Solution

1. Preculture the bacteria overnight by picking up a colony in 4ml LB with antibiotic.


2. Culture the bacteria in 30 ml LB by adding 1/100 of precultured broth , 1/2000 kanamycin, 1/4000 chloramphenicol to log phase( about 3 hr).


3. Centrifuge them in 22℃ ,3200xg for 5 minutes , then refresh by the M9 medium with 4%xylose and 0.1%LB.


4. Culture the bacteria for 24 hr in both O2 and 5%CO2 incubator , and test the O.D. value at every 6 hours.