A． Standard IGEM method

1.The composition of LB medium for E. coli
1)Liquid medium (1L)
NaCl 10g
Tryptone 10g
Yeast extract 5g
Distilled water 1000ml
2)Solid medium
NaCl 10g
Tryptone 10g
Yeast extract 5g
Distilled water 1000ml
Agar powder 10~20g
Sterilize the mixture in autoclave at 121℃ for 30 minutes.

2.The preparation of competent E.coli cell (DH5α)
1)pick one colony and put it in 3~5ml LB media , overnight at 37℃
2)Transfer the centrifugal sediment into 100ml LB liquid media
3)Grow the cuLture at 37℃(250rpm), until OD600 = 0.4 (2-3h)
4)Place cuLture on ice for 10 minutes
5)Centrifuge cuLture at 4℃ for 10 minutes at 4,000 rpm Subsequent resuspensions is done in the same tube. CuLture remains cold for the rest of procedure. Place tubes on ice and resuspend the cuLture the on ice
6)Remove the media and resuspend cells in 10ml cold 0.1 M CaCl2 and incubate it on ice for 30 minutes
7)Centrifuge it at 4℃ for 10 minutes at 3,500 rpm
8)Remove the supernatant and resuspend cells (by pipetting) in 4 ml cold 0.1 M CaCl2 containing 15% glycerol. Transfer 100uL the mixture into 1.5 ml centrifuge tubes placed on ice. Cells are stored at -80℃ and can be used for transformation in 6 months

3.Transformation
1)Thaw the competent cells on ice
2)Add 50 uL of thawed competent cells into pre-chilled 1.5ml tube
3)Add the total ligation plasmid into the 1.5 ml tube. Overturn gently the tube a few times. Make sure that the competent cells is always on ice
4)Close the tubes and incubate the mixture on ice for 30 minutes
5)Heat shock the cells by water bath at 42℃ for 45 seconds
6)Place the cells on ice for 1 minutes
7)Add 400 uL LB liquid media into the tubes
8)Incubate the cells at 37℃, 110rpm for 1 hour

4.Restriction enzyme reaction system
1)formula
2)Mix gently and centrifuge it for a few seconds
3)Incubate it at 37℃ for 5 minutes
The combination of double enzyme digestion are as following:
FD EcoRⅠ、FD Pst Ⅰ
FD XbaⅠ、FD Pst Ⅰ
FD EcoRⅠ、FD Spe Ⅰ
FD SpeⅠ、FD Pst Ⅰ
FD EcoR Ⅰ、FD Xba Ⅰ
The enzymes using in the experiments are all from Thermo company.

5. Ligation system reaction
1)Prepare the following reaction system:
2)Incubate it more than 1 hour at 22 ℃ water bath

6. PCR reaction
1)Prepare the following reaction system:
2)The PCR reaction setting:

7. Agarose Electrophoresis
1)Prepare a piece of 1% weight-to-volume agarose and add SYBR dye or ethidium bromide
2)Place the agarose in the apparatus rig with the wells facing the negative electrode (black-colored)
3)Fill the rig with 1x TAE buffer
4)Load 5 uL 1 kb marker
5)Add 1 uL of 6x buffer to 5 uL DNA sample. Load the mixture
6)Run at 120V

8. Purification of DNA
1)Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice
2)Weigh the gel slice and add 3 volumes of gel lysis buffer to every 1 volume of gel(100mg = 100 uL)
3)Wait 2 minutes
4)Centrifuge it at 12,000 rpm for 1 minute
5)Discard the flow-through and repeat Step 4 until all sample has passed through the column
6)Add 700 uL rinse buffer into the column, then centrifuge it at 12,000 rpm for 1 minute
7)Discard the flow-through and add 500 uL rinse buffer into the column, then centrifuge it at 12,000 rpm for 1 minute
8)Discard the flow-through and centrifuge the column at 12,000 for 2 minutes
9)Transfer the column to a new centrifuge tube. Open the tube at 37℃ for a few minutes to volatilize the alcohol
10)Add 40 uL elution buffer to the center of the column and wait at least 2 minutes
11)Centrifuge it at 12,000 rpm for 1 minutes

B. Gibson Protocol

1. Extend each fragment using the PCR protocols below
2. Make up the Assembly Mix with the PCR products
3. Incubate the Assembly Mix for 1 hour at 50°C
4. Purify/transform

Assembly Mix Volume
Gibson Master Mix 15 µl
Fragment with same mole rate 5 µl
Total 20 µl

Gibson Master Mix Volume
Taq ligase (40u/µl) 50 µl
5x isothermal buffer 100 µl
T5 exonuclease (1u/µl) 2 µl
Phusion polymerase (2u/µl) 6.25 µl
Nuclease-free water 216.75 µl
Total 375 µl

5x isothermal buffer Volume
25% PEG-8000 0.75g
500 mM Tris-HCl pH 7.5 1500 µl
50mM MgCl2 75 µl
50mM DTT 150 µl
1mM dATP 30 µl
1mM dTTP 30 µl
1mM dCTP 30 µl
1mM dGTP 30 µl
5mM NAD 300 µl
Nuclease-free water ...
Total 3000 µl

C. Integration od a linearized plasmid into the yeast genome

Material:
o       DNA mix: 20ul of solution containing a linearized plasmid;
o       LiAc mix, Salmon Sperm DNA, and PEG mix;
o       DMSO, YPD solution, and agar plates (SD-URA or SD-TRP);
o       Cuvettes (OD measurement) and sterile beads (cell plating).

Method (ALWAYS STERILE CONDITIONS):
1st day:
-        grow yeast cells overnight into YPD

2nd day:
-        Fast method:
       Measure cell culture OD;
       If OD > 10 pour 500ul cell solution into 20ml YPD. Use glass flasks with baffles.
       Grow cells at 30 Celsius degrees and 130 RPM for 5-6 hours.

-        Pour the 20 ml cell solution into one 50 ml Greiner tube.
-        Centrifuge at max 1000 RCF for 5 minutes at room temperature.
-        Throw away the supernatant.
-        Resuspend with 5 ml of sterile water by inverting the tube gently.
-        Centrifuge at 3000 RCF for 5 min at room temperature.
-        Throw away the supernatant.
-        Resuspend with 200 ml of LiAc mix.
-        Transfer 100 ul of cells solution into the 1.5 ml eppendorf containing the 20ul DNA mix.
-        Add 10 l of Salmon Sperm DNA—some salmon sperm DNA might require to be boiled at 95 Celsius degree (only the salmon sperm DNA) for 5 min every third time you use it. Let it cool down at room temperature.
-        Add 600 l of PEG mix.
-        Put the plates into the incubator at 30 Celsius degrees.
-        Incubate the 1.5 ml eppendorf on the wheel for 30 min at low speed ~0.1.
-        Add 73 ul DMSO.
-        Make a heat shock by incubating the eppendorf on a small shaker for 15 minute at 42 Celsius degrees and high speed (1250 RPM).
-        Centrifuge at max 0.8 RCF at room temperature for 2 min.
-        Suck off the supernatant with a pipette.
-        Resuspend with 300 ul YPD. Mix the solution with a pipette.
-        Incubate for 20 min on the wheel (at low speed again).
-        Depose 300 ul on the corresponding plate.
-        Spread the cell solution on the plate with sterile beads.
-        Incubate for 2 nights in the incubator at 30 Celsius degrees.