Team:Shanghai City United/Experiments

Shanghai_City_United

EXPERIMENTS

1. Preparation of LB medium

Materials:

Yeast extract 5g/L

Tryptone 10g/L

NaCl 10g/L

Agar(only for solid medium)1.5%

H2O (as solvent) 1000ml

Totally 1000ml (600ml for liquid medium, 400ml for solid medium)

Steps:

1. Weighing 5 gram yeast extract

2. Preparing 1000ml, and pouring into a huge measuring cylinder

3. Adding the yeast extract into water

4. Weighing 10 gram Tryptone

5. Adding the tryptone into water

6. Weighing 10 gram sodium chlorine

7. Adding sodium chlorine into water

8. Stirring the solution

9. Preparing 5 flasks 3 of them are 250ml, and 2 are 500ml

10. Adding 150ml solution into the 250ml flasks, and adding 300ml solution into 500ml solution

11. For solid medium, adding the agar into the bottom of the flask first, and than pouring the solution into it.

12. Use the membrane to seal the flask( double filter, 4 pores for air recycle)

13. Put the flask into the High-Pressure Steam Sterilization Pot for sterilization(0 is standard pressure 1atm)(condition: 0-0.4, 120 centigrade 30 minute)

2. PCR amplification

Materials:

Primer-F 1μL

Primer-R 1μL

2X Prime Star Max(DNA polymerase ,Buffer, dNTP) 50μL dillute to 1/2 25μL

Template 1μL

ddH2O 22μL

In the PCR machine:

98℃ 5m pre-denaturation (break the hydrogen bond)

98℃ 15s break the hydrogen bond

55℃ 15s primer connect to the brands

72℃ 5s/1k bp extend the DNA strands(the temperature which the enzyme most active)

Repeat these three steps 30 times

72℃ 3m let the DNA strands completely extend sufficiently

4℃ preserve the DNA

Process:

1. Primer 5.00 n mol diluter in water

2. 500μL water add into Primer-F

3. Split 200μLPrimer-F

4. Adding template into 900μLwater(dilute)

5. Adding 900μLwater into template

6. Adding 22μL ddH2O into the PCR tube

7. Adding 25μL Primer Star Max into the PCR tube

8. Adding 2 primers

9. Adding template

10. Marked the number (determine)

11. Put in the PCR machine(shut the top cover avoid the liquid evaporate)

12. Setting the program has written above.

13. Waiting for the process finish

14. Preserve the DNA.

3. Running nucleic acid electrophoresis

Mechanism: DNA is negative charge, so it will be attract to the positive direction. Also, different DNA contains different number of base pair and shape. The speed of DNA are different either. We can compare the model DNA(500bp) with ours product after PCR to determine whether our products are that one we need.

A. Nucleic Acid Electrophoresis Gel

Materials:

1. Agarose 1%

2. TAE buffer 100ml

3. Nucleic acid dye 10μL

4. Marker

5. DNA from PCR

Process:

1. Mix the agarose and 100ml TAE buffer

2. Heated in microwave about 3 min until all the agarose melt.

3. Add Nucleic acid dye into the solution.(double glove avoid the dye contact skin)

4. Pour the heated solution into the mold tank and plate and wait it to solid

5. Take the gel(use knife around the gel and pick it up)

6. Put the gel in the bottom of the tank

7. Pour TAE buffer into the tank until it cover the gel

8. Add the marker into the first ole of the gel(do not penetrate the gel)

9. Add Loading buffer into PCR tube(let the DNA get color)

10. Add other DNA into the gel.

11. Clean the surrounding of the tank and cover it

12. Energized it with 120V(there are bubbles going out from the liquid)

13. Waiting about 15 min

14. Observe the traits of the DNA under the violet light.

4. Measuring OD600

Mechanism: The light absorption of bacteria is used to measure the concentration of bacterial culture medium, so as to estimate the growth of bacteria. It is usually used to refer to the density of bacterial cells.
Experimental Procedure

1. To measure the concentration, we use Nanodrop by measuring light absorption value.

2. Use 2ul Micro pipette with ddH2O to clean the water on the sampling table, wipe the sweat with dust-free paper, repeat three times.

3. Drop 2ul DNA sample on the detection platform and the internal light penetrate the DNA sample.( We have 100% internal light, if the metering wall shows 20% light, then we will know that the DNA sample absorb 80% light.) (A260/A230→calculation of concentration, A260/A280→calculation of purity—1.8 is close to pure)

4. After the experiment, the ddH2O cleaning process should be repeated three times again.

5. Building Plasmid (Double Digest&T4 connection)

Lactobacillus’s route:

1. Carry on processing the previous E. coli that carries XynA, select white strains that express lac ab enzyme.

2. Injecting the selected strains into LB nutrient solution and Amp antibiotic solution.

3. Extracting the plasmids from PMD19-T—XynA. (Which has been mentioned in detail later)

4. Double digestion for different biological parts that eventually constructed the final plasmid:

First time:

Using restriction enzymes to cut different parts that serves for the connections. Using NheI and HindIII to cut the part of XynA; Using BamHI and NheI to cut the part of PUS; Using BamHI and HindIII to cut the part of PSIP403. Three cutted parts need to run the nucleic acid electrophoresis, getting the target length around 500bp for XynA; 300bp for PUS and 6kbp of PSIP403. (Notice worthily, by using the formal strategies on double digestion, we failed on the step of transplanting the building plasmids into the Lactobacillus. Excluding the effect of erythromycin, we’ve concluded that two restriction sites on the parts may cut off the segment of the genetic expression for ori---a codon initiator for the functioning of this plasmid inside the Lactobacillus.)

Second time:

By first using PCR techniques for amplifying the restriction site fragment, we’ve changed one of our restriction enzymes from BamHI to EcoRI. (Details for the solutions are provided bellow)

5. T4 connecting system

(PSIP403：PUS : XynA=1:1:1)

10X T4 Ligase buffer 1μL

psiP403 0.5μL

Pus 5.5μL

XynA 2μL

T4Ligase 1μL

Solutions above need to be mixed in a testing tube to react for at least 30 minutes under the surrounding temperature of 6 degrees.

6. Cultivating E.coli

Lab procedure:

1. Process the E.coli DH5α with Cacl2 to depolarize the membrane of the cell and allow access of exterior DNA.

2. Extract 50ul of E.coli DH5α from stored glycerin. And transfer into a 2ml Eppendorf tube.

3. Extract and add 5ul of T4 Polymerase (XynA) to the Eppendorf tube.

4. Unfreeze the minus eighty degrees’ cells on ice for 30 minutes. (In order to allow DNAs to do free diffuse)

5. Water bath the Eppendorf tube to heat shock the E.coli for 90 seconds to let the plasmids absorb through the openings on the cell.

6. Place the Eppendorf tube on ice for 30 minutes to entirely close the openings on the cell and recovering the cell from competent.

7. 895ul of LB liquid culture medium were added under the sterile operating table as nutrients.

8. The Eppendorf tube then put in a beaker and placed in a shaking incubator for 30 minutes. (240rpm)

9. Then the Eppendorf tube was centrifuged for 30 seconds. (12000 times/min, centrifugal force: 13800)

10. Solid LB culture medium was added into petri dish with a final concentration of 100ug/ml Amp filtrated stock; 20ug/ml IPTG filtrated stock and 20ug/ml X-Gel filtrated stock.

11. Burned the micro pipette tips with the alcohol burner and placed upside down till the top of tip smoothen.

12. Extract 800ul of bacteria from the Eppendorf tube and safe the rest of it.

13. 50ul of droplets should gently drop in the center of the petri dish and rub back and forth to coated the plate.

14. Put the coated plate upside down into the incubator and incubate overnight at 37℃.

7.Plasmid extraction（PMD19-XynA)

1. Add E.coli DH5 α/PSIP403-PUS-XynA into 4ml LB solution and put it into shaking incubator. (37℃, 240rmp, 16h)

2. Add 250ul buffer SP1 and blow and mix well. (SP1: Glucose- form a suspension with suspended bacteria; RNase- degrade RNA; Buffer- provide the environment)

3. Add 250ul buffer SP2, reverse it 5 to 10 times. SP2: NaOH–Cleavage cell wall, slightly clarified liquid, DNA&RNA release from the cell; metal chelating agent–wipe off the metal ion

4. Add 350ul buffer SP3, quickly invert and mix. SP3: SDS (protein denaturant)–Let the protein denature; CH3COOK–Neutralize PH (→NaOH), K→ The metal ions are turned into water-insoluble salts and precipitated

5. Centrifuged it in a centrifuge. (5min, 12000 times/min, centrifugal force: 13800) Then we get: Sediment: Protein, metal ions (some of), genome (wrap around the protein)/Supernatant liquid: plasmid DNA, metal ions (some of ), residual protein (some dissolve in the water)

6. Add 500ul buffer S on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom. (Column can be activated and balanced./ Wash the silicate fiber membrane.)

7. Add 800ul supernatant liquid on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom. (Plasmid DNA can be attached to the membrane.)

8. Add 500ul buffer DW1 on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800)Pour out the waste liquid at the bottom. (Remove residual protein)

9. [Add 500ul wash buffer(C2H6O) on the adsorption column. Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom.] *2 （Cleaning residue, like salt, metal, ions...)

10. Put it in a centrifuge without adding any liquid. (30s, 12000 times/min, centrifugal force: 13800) (Centrifuge the wash buffer)

11. Move the column to a new 1.5ml tube, open the lid and let stand for 1min. (C2H6O volatulization)

12. Add 50ul ddH2O (50℃ hot water) (Elution buffer cannot be used, Elution buffer--Tris-HCl×) Centrifuged it in a centrifuge (60s, 12000 times/min, centrifugal force: 13800) (Eluting plasmid DNA)

13. Throw the column into the lab pollution bin. (Remaining: plasmid DNA and ddH2O)

8. Running SDS PAGE protein electrophoresis

Make SDS PAGE gel

1. 2.7ml lower layer solution, 2.7ml lower layer buffer, 60ul improved coagulant. (Blow and mix. It will set in 5-10 minutes)

2. Slot: Add water and see if the liquid level drops. It is set when the liquid level doesn’t drop.

3. Add 1000ul for each time (The distance between the liquid level and the short glass plate is 0.5cm longer than the comb length.)

4. Add some water to see if the liquid is set.

5. 0.75ml upper glue solution, 0.75ml red upper buffer, 15ul coagulant, mix, pour in, and insert the comb.

Running

Mechanism: Spacer gel: superstratum, small concentration, large pore. It helps the protein to press into a line at the boundary between the upper and lower layers. Separation gel: substratum, large concentration, small pore. From here on, the small ones swim faster than the big ones.

1. Power up and start running（120v/30min)

2. We can stop when the blue line reaches ths bottom.

Coomassie brilliant blue staining

1. Pour CBB into a box to cover the gel in it. Rapid dyeing: microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB.

2. [Pour CBB distaining solution. Rapid wash the dye: microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB distaining solution.] *3-4

9. Measuring the enzyme activity

Xylan Liquid (50mL):

0.25g of xylan (5g/L)

Add ddH2O until 50mL

Reaction Medium (1mL):

·Water bath--37℃--15min, 90℃--5min, add DNS--2ml, 90℃--8min, add ddH2O 7ml (the capacity to 10mL)

·Let it cool to room temperature.

·Measuring 540nm light absorption value（The procedure is the same as that for OD600）