Difference between revisions of "Team:CIEI-China/Protocols"

LB

Materials	Amount
Yeast Extract	5 g
Tryptone>	10 g
NaCl	10 g
ddH2O	To 1 L
Ampicillin (20 μg/mL in H2O)	200 μL

1.Add the materials above in a conical flask.

2.Shake the conical flask till all the materials were dissolved.

3.Autoclave the conical flask at 15 psi for 15 min to sterilize on liquid cycle.

LB agar

Materials	Amount
Yeast Extract	5 g
Tryptone	10 g
NaCl	10 g
Agar	15 g
ddH2O	To 1 L

1.Add the materials above in a conical flask.

2.Shake the conical flask till all the materials were dissolved.

3.Autoclave the conical flask at 15 psi for 15 min to sterilize on a liquid cycle.

4.Then swirl the conical flask gently. After it cool down to 50~60°C, add antibiotics, Ampicillin. Swirl the flask to mix them up.

5.Pour the LB agar in the conical flask on plates before it solidified.

1.Prepare the reaction mix in PCR tube:

Template DNA	1 μL
Enzyme (prime STAR)	1 μL
Forward Primer	2 μL
Reverse Primer	2 μL
Easy Jag Mix	25 μL
ddH2O	19 μL

Note: We used two types of template DNA with the volume of 1.5 μL

2.Set the program of thermal cycle:

1x	95˚C	5 min
30x	95˚C	30 s
30x	55˚C>	30 s
30x	72˚C	1.5 min
1x	72˚C	10 min
1x	16˚C	15 min

BL Buffer	500 μL
PB Buffer	250 μL
PW Buffer (with 100% ethyl alcohol)	600 μL
EB Buffer	40 μL
PCR product system	50 μL

1. Add BL buffer into spin column CB2 (with collection tube of 2 mL) and centrifuge it for 1 min at 12000 rpm. Remove the discard from the collection tube.

2. Add the PCR product and PB buffer in the CB2 and mix them evenly. (Notice: If the PCR product system is XμL, add 5XμL PB buffer)

3. Leave the mixture for 2 min and then centrifuge it for 1 min at 12000 rpm. Remove the discard from the collection tube. (Notice: The max volume of column is 800 μL, if the sample volume is more than 800 μL, you can repeat step 3)

4. Add 600μl of Buffer PW to column CB2. Centrifuge it at 12,000 rpm for 30-60 sec in a microfuge. Pour the discard solution from the tubes; put columns CB2 back to the tube. Store the tube at room temperature for 2-5 min.

5. Repeat step 4 once.

6. Centrifuge the tube at 12,000 rpm for 2 min, pour the discard solution. Store the tube at room temperature for 5 min, open the tube to leave the column as dry as possible. Take the DNA solution in the collection tube to carry out gel electrophoresis.

10×Loading Buffer	2.5 μL
Ligase	2.5 μL
AD plasmid fragment	10 μL
BD SOS fragment	10 μL
Total	25 μL

1.Mix the materials above in a 0.2 mL microfuge tube.

2.Incubate the mixture at 4 C overnight or at 16 C for 2-3 hrs.

Template	1400 μL*2
P1	1 μL
P2	2 μL
P3	2 μL
Washing Buffer	19 μL
Elution Buffer	1 μL

Note: Templates include carrier with target gene (gltB, ScTPS1 and SpTPS1)

1.Pour 1400 μL of the culture into a microfuge tube. Centrifuge the mixture at 12,000 rpm for 1 min in a microfuge. Pour the discard solution, leaving the leftover as dry as possible.

2.Repeat step 1 once.

3.Add 400 μL solutions P1 into the microfuge tube, wait until all deposition dissolves.

4.Add 400 μL solutions P2 to the microfuge tube; mix the content by inverting the tube rapidly for five times.

5.Add 560 μL solutions P3 to the mixture, wait until all deposition dissolves.

6.Centrifuge the microfuge at 12,000 rpm for 10 min. Transfer the supernatant into a fresh column which is in a tube.

7.Centrifuge the supernatant at 12,000 rpm for 1 min in a microfuge. Discard the solution in tube.

8.Add 700 μL Washing buffer (PB) to the column, then centrifuge the mixture at 12,000 rpm for 1 min. Pour the discard solution in tube.

9.Add 500 μL Washing buffer (PB) to the column, then centrifuge the mixture at 12,000 rpm for 1 min. Pour the discard solution in tube.

10.Centrifuge the tube at 12,000 rpm for 2 min, then open the tube and dry the column.

11.Transfer the column to a fresh microfuge tube, then add 80 μL Elution buffer (EB), store it at room temperature for 5 min.

12.Centrifuge the microfuge tube at 12,000 rpm for 1 min, and then discard the column.

13.Collect the leftover in tube after centrifuge, which was the plasmid DNA

1.Mix 0.25 g of agarose with 25 g of TAE solution, and then heat the mixture until the agarose is fully dissolved.

2.Pour the mixture into a square mould. Plug in the comb, and wait until the mixture solidifies.

3.Put the gel into the electrophoresis apparatus, and then fill it with TAE solution.

4.Add 1 μL of loading buffer and 1 μL of SYBR Gold into 5 μL of the sample.

5.Load the sample and marker into the gel. Connect two sides with the electric source. Set the voltage at 120 V and then wait for 30-40 min.

6.Take the gel out, and observe the results under UV transillumination.

Separating gel (12%)

dd water	3.92 mL
30% Acr-Bis	4.80 mL
1.5M Tris (pH 8.8)	3.04 mL
10% SDS	120.00 μL
10% ammonium persulfate	120.00 μL
TEMED	4.8 μL

Note: waiting 40min until it become solid

Stacking gel (5%)

dd water	2.04 mL
30% Acr-Bis	0.51 mL
1.0M Tris (pH 6.8)	0.375 mL
10% SDS	30 μL
10% ammonium persulfate	30 μL
TEMED	3 μL

Note: waiting 40min until it become solid

1.Assemble the gel into the electrophoresis apparatus, and then fill the inner chamber with buffer.

2.Remove the comb carefully, then load the sample and marker. The volume of each specimen is 10-15 μL.

3.Fill the bottom of the outer chamber from the top of the gel box.

4.Attach the power supply, set the voltage at 80 V, after 20 min until the sample move into the separating gel, and then change it into 100 V. When the bromophenol blue reach the bottom, stop it.

YPD agar

Materials	Amount
1% Yeast Extract	10 g
2% Peptone	20 g
2% Dextrose	100ml
Agar	20 g
ddH2O	To 1 L
salinity	0%, 0.5%, 0.75%, 1%, 1.25%

1.Add the materials above in a conical flask (except the dextrose).

2.Shake the conical flask till all the materials were dissolved.

3.Autoclave the conical flask at 121°C for 20 min to sterilize on a liquid cycle.

4.Then swirl the conical flask gently. After it cool down to 50~60°C, add dextrose. Swirl the flask to mix them up.

5.Pour the LB agar in the conical flask on plates before it solidified.

6.Culture yeast that with our target gene and the yeast without our target gene. Couple days later, then test the survival rate of both kinds of yeasts.

1.Dry the materials thoroughly in the oven.

2.Grind the dried materials into powder; weigh them, then add five times volume (mass-to-volume ratio) of boiling distilled water and fully mix them. (For example, 5g dried powder require adding 25mL boiling distilled water.) Extract the supernatant after standing or filtration.

3. Measure conductivity of the supernatant by conductivity meter and convert the data into salt concentration. (At 25̊C, based on the mass/volume ratio of 1:5, the salt concentration is calculated as the conductivity multiplied by 0.064.)

Principle: according to the fact that the salt concentration is proportional to the conductivity, the salt concentration can be calculated by measuring the conductivity of the samples.