Revision as of 01:13, 13 October 2016

Team SUSTC-Shenzhen

Biobricks

Notebook

8 Chromoprotein

Plasmid Construction

ALL ABBREVIATIONS USED:

Name	Number	Index	Position	Backbone	Length
Strong promoter		BBa_J23100	4-17-D	BBa_J61002	35
Weak promoter		BBa_J23106	4-17-P	BBa_J61002	35
Strong RBS	③	BBa_B0034	4-1-N	pSB1A2	12
Weak RBS	④	BBa_B0031	2-2-H	pSB1C3	14
Terminator	⑤	BBa_B0015	3-3-F	pSB1C3	129
eforRed	⑥	BBa_K592012	6-15-I	pSB1C3	681
gfasPurple	⑦	BBa_K1033919	6-9-K	pSB1C3	669
FwYellow	⑧	BBa_K1033910	4-6-K	pSB1C3	714
SpisPink	⑨	BBa_K1033932	6-11-k	pSB1C3	678
①+③	⑩	BBa_K880005	2-3-F	pSB1C3	55
Medium promoter+④	11	BBa_K608007	1-5-G	pSB1C3	57

7.22

Transformation of ①, ③, ⑤

Time: 20:30

Handler: Liao Weiduo

Procedure: IGEM Protocols

1. Thaw competent cells on ice.

2. Punch a hole into the corresponding hole in the hole in 2016 kit plate by a 10 ul pipette tip. Adding 10ul ddH₂O to the hole and pipette up and down for several times. Let sit for several minutes to make sure the dyed DNA is fully resuspended.

3. Pipette 50ul of competent cells into 1.5ml tube.

4. Pipette 1ul of resuspended DNA into 1.5ml tube.

5. Pipette 1ul of ddH₂O as a control into 1.5ml tube.

6. Close 1.5ml tubes, incubate on ice for 30 min.

7. Heat shock tubes at 42℃ for 1 min.

8. Incubate on ice for 5 min.

9. Pipette 200ul SOC media to each transformation.

10. Incubate at 37℃ for 2 hours, 220rpm.

11. Pipette each transformation on petri plates for a 100ul, and spread with sterilized spreader immediately.

12. Incubate transformations overnight at 37℃.

7.23

Picking single colonies of ①, ③, ⑤

Time: 16:00

Handler: Liao Weiduo

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
Incubate overnight at 37℃, 220rpm.

7.24

Plasmid extraction of ①, ③, ⑤ A and B

Time: 9:00

Handler: Liao Weiduo

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Add 2ml LB medium to a 2ml microcentrifuge tube.
Centrifuge at 10,000 g for 1 minute at roonm temperature.
Discard the supernatant.
Repeat Step1-3 until 4ml sample has been transferred to the 2ml tube.
Add 250ul Solution I/RNase A. Vortex to mix thoroughly.
Add 250ul Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
Add 350ul Solution III. Immediately invert several times until a flocculent white precipitate forms.
Centrifuge at maximum speed (>13,000 g) for 10 minutes.
Insert a HiBind DNA Mini Column into a 2ml Collection Tube.
Transfer the cleared supernatant (~830ul)from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column.
Centrifuge at maximum speed for 1 minute.
Discard the filtrate and reuse the collection tube.
Add 500ul HBC Buffer. (HBC Buffer must be diluted with isopropanol before use)
Centrifuge at maximum speed for 1 min.
Discard the filtrate and reuse collection tube.
Add 700ul DNA Wash Buffer. (DNA Wash Buffer must be diluted with 100% ethanol prior to use.)
Centrifuge at maximum speed for 1 min.
Discard the filtrate and reuse the collection tube.
Repeat Steps 16-18.
Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
Transfer the HiBind DNA Mini Column to a clean 1.5ml microcentrifuge tube.
Add 80ul ddH₂O.
Let sit at room temperature for 2 min.
Centrifuge at maximum speed for 1 min.

Results:

‘’’Concentration:’’’ ①A: 245.9 ng/ul 1.84; ①B: 340.5 ng/ul 1.84; ③A: 116.2 ng/ul 1.65; ③B: 102.1 ng/ul 1.77; ⑤A: 151.4 ng/ul 1.80; ⑤B: 68.0 ng/ul 1.84;

7.25

Transformation of ②,④,⑥,⑦,⑧,⑨

Time: 0:00

Handler: Tang Shiqiang

Procedure: IGEM Protocols

Picking single colonies of ②,④,⑥,⑦,⑧,⑨

Time: 20:00

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, α and β.
Incubate overnight at 37℃, 220rpm.

7.26

Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group α

Time: 9:40

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ②α: 369.8 ng/ul; ④α: 142.4 ng/ul; ⑥α: 116.5 ng/ul; ⑦α: 92 ng/ul; ⑧α: 157.4 ng/ul; ⑨α: 168.3 ng/ul.

‘’’ Note: ’’’ ⑧ has a red colony.

Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group β

Time: 14:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ②β: 496.9 ng/ul 1.86; ④β: 256.4 ng/ul 1.85; ⑥β: 220.3 ng/ul 1.85; ⑦β: 225.7 ng/ul 1.85; ⑧β: 304.6 ng/ul 1.85; ⑨β: 352.2 ng/ul 1.84.

‘’’ Note: ‘’’ ⑧ has a red colony.

7.27

Competent Cell Test

Time: 16:00

Handler: Lu Shixin

Procedure: IGEM Protocols

Spin down the DNA tubes form the Competent Cell Test Kit to collect all of the DNA into the bottom of each tube prior to use.
Thaw competent cells on ice.
Pipet 50ul of competent cells into each tube.
Pipet 1ul of DNA into each microcentrifuge tube.
Incubate on ice for 30 min.
Heat shock at 42℃ for 1 min.
Incubate on ice for 5 min.
Add 200ul SOC media, incubate at 37℃ for 2h.
Pipet 20ul from each tube onto the appropriate plate, and spread it.
Incubate at 37℃ overnight, approximately 16 h.

Enzyme Digestion and Go Gel Electrophoresis Test of ②,④,⑥,⑦,⑧,⑨

Time: 20:00

Handler: Xiao Tianyao

Procedure:

1. Make enzyme cutting system solution.

	②α	④α	⑥α	⑦α	⑧α	⑨α
DNA(400ng)	1ul	2ul	2ul	2ul	1.2ul	1.2ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul
EcoRI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
PstI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	16.6ul	15.6ul	15.6ul	15.6ul	16.4ul	16.4ul
Total	20ul	20ul	20ul	20ul	20ul	20ul

	②β	④β	⑥β	⑦β	⑧β	⑨β
DNA(400ng)	1ul	2ul	2ul	2ul	1.2ul	1.2ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul
EcoRI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
PstI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	16.6ul	15.6ul	15.6ul	15.6ul	16.4ul	16.4ul
Total	20ul	20ul	20ul	20ul	20ul	20ul

Incubate at 37℃ for 30 min.
Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.

Results:

figures show below:

SUSTech Shenzhen-9BCCBDAD-C55F-46B0-B96C-D880F0C7F660.png

‘’’Comment:’’’ ⑧ has some problem, and we will redo it.

7.28

Competent Cell Test Colonies Counting

Time: 13:00

Handler: Lu Shixin

Result:

SUSTech Shenzhen-C89646A9-AD79-4481-95BB-E54BBC7C1B87.png

Figure 1. Water control

SUSTech Shenzhen-D6D86280-E0BE-43CE-A096-8C835F4D8426.png

Figure 2. 0.5 pg/ul

SUSTech Shenzhen-9A343E4A-0565-47D6-BD89-A9FA00F5EEA2.png

Figure 3. 5 pg/ul Figure 4. 10 pg/ul

SUSTech Shenzhen-FBC625A4-EE60-428A-91AA-213E132DDDFC.png

Figure 5. 20 pg/ul Figure6. 50 pg/ul

And all the colonies are red under UV light.

7.29

Transformation of ⑧,⑩，11

Time: 16:00

Handler: Lu Shixin, Tang Shiqiang

Procedure: IGEM Transformation Protocol

Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑧,⑨

Time: 20:00

Handler: LuShixin, Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑥	⑦	⑧	⑨
DNA（500ng）	2.5ul	2.5ul	2.0ul	1.5ul
Cutsmart buffer	2.5ul	2.5ul	2.5ul	2.5ul
EcoRI-HF	0.5ul	0.5ul	0.5ul	0.5ul
SpeI-HF	0.5ul	0.5ul	0.5ul	0.5ul
ddH2O	19ul	19ul	19.5ul	20ul
Total	25ul	25ul	25ul	25ul

	⑤
DNA (500ng)	8ul
Cutsmart buffer	2.5ul
EcoRI-HF	0.5ul
XbaI	0.5ul
ddH2O	16.5ul
Total	25ul

Incubate at 37℃ overnight. (Start from 21:20)

7.30

Picking single colonies of ⑧, ⑩, 11

Time: 9:00

Handler: Lu Shixin

Procedure:

Pick single colonies, adding 1ml LB with corresponding antibiotics in 1.5ml EP tube, each plate we pick 2 single colonies, α and β.
Incubate for 2h at 37℃.
Transfer 1ml LB from 1.5ml EP tube to 14ml bacteria shaking tube, and add 5ml corresponding antibiotics LB.
Incubate at 37℃ for 10h, 220rpm.

Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑧,⑨

Time: 9:00

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.
Excise the DNA fragment of interest.
Determine the volume of the gel slice by weighing it in a clean 1.5ml EP tube.
Add 1 volume Binding Buffer (XP2).
Incubate at 50-60℃ for 7 min or until the gel has completely melted. Vortex or shake the tube every 2-3 min.
Insert a HiBind DNA Mini Column in a 2ml Collection Tube.
Add no more than 700ul DNA/agarose solution from Step 7 to the HiBind DNA Mini Column.
Centrifuge at 10,000 g for 1 min at room temperature.
Discard the filtrate and reuse collection tube.
Repeat Steps 9-11 until all of the sample has been transferred to the column.
Add 300ul Binding Buffer(XP2).
Centrifuge at maximum speed (>13,000 g) for 1 min at room temperature.
Discard the filtrate and reuse collection tube.
Add 700ul SPW Wash Buffer. (SPW Wash Buffer must be diluted with 100% ethanol prior to use.)
Centrifuge at maximum speed for 1 min at room temperature.
Discard the filtrate and reuse collection tube.
Repeat Steps 16-18.
Centrifuge the empty HiBind DNA Mini Column for 2 min at maximum speed to dry the column matrix.
Transfer the HiBind DNA Mini Column to a clean 1.5ml EP tube.
Add 15-30ul ddH₂O.
Let sit at room temperature for 2 min.
Centrifuge at maximum speed for 1 min.

Results：

SUSTech Shenzhen-4E6C74F1-3B4C-4F1A-B283-6751068590D8.png

All the concentration of gel extraction are below 10 ng/ul. So we decide to repeat enzyme digestion with more DNA.

Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑨ (Redo)

Time: 18:00

Handler: Lu Shixin

Procedure:

Make enzyme cutting system solution.

	⑥	⑦	⑨
DNA（>4ug）	21.5ul	21.5ul	21.5ul
Cutsmart buffer	2.5ul	2.5ul	2.5ul
EcoRI-HF	0.5ul	0.5ul	0.5ul
SpeI-HF	0.5ul	0.5ul	0.5ul
ddH₂O	0ul	0ul	0ul
Total	25ul	25ul	25ul

	⑤
DNA (2ug)	21.5ul
Cutsmart buffer	2.5ul
EcoRI-HF	0.5ul
XbaI	0.5ul
ddH₂O	0ul
Total	25ul

Incubate at 37℃ for 3h. (Start from 18:40)

Plasmid extraction of ⑧,⑩,11

Time: 21:00

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

The length of them are correct.

Concentration: ⑧: 130.2 ng/ul; ⑩: 147.0 ng/ul; 11: 93.3 ng/ul.

Enzyme Digestion for Gel Extraction of ⑧,⑩,11

Time: 22:30

Handler: Lu Shixin

Procedure:

Make enzyme cutting system solution.

	⑩	11
DNA（>1.5ug）	11.5ul	21.5ul
Cutsmart buffer	2.5ul	2.5ul
SpeI-HF	0.5ul	0.5ul
PstI-HF	0.5ul	0.5ul
ddH₂O	10ul	0ul
Total	25ul	25ul

	⑧
DNA (0.5ug)	4ul
Cutsmart buffer	2.5ul
EcoRI-HF	0.5ul
XbaI	0.5ul
ddH₂O	17.5ul
Total	25ul

Incubate at 37℃ overnight. (Start from 22:40)

Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑨ (Redo)

Time: 22:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

Gel extraction concentration: ⑤: 15.3 ng/ul; ⑥: 36.3 ng/ul; ⑦: 36.8 ng/ul; ⑨: 26.9 ng/ul.

7.31

Ligation of ⑥+⑤, ⑦+⑤, ⑨+⑤

Time: 0:00

Handler: Tang Shiqiang

Procedure:

Make ligation system solution.

	⑥+⑤	⑦+⑤	⑨+⑤
Vector (50ng)	3.3ul	3.3ul	3.3ul
Insert	1.4ul	1.4ul	1.9ul
ddH₂O	5.3ul	5.3ul	4.8ul
2x Quick ligase buffer	10ul	10ul	10ul
Quick ligase	1ul	1ul	1ul
Total	21ul	21ul	21ul

Incubate at room temperature for 5 min.
Take 2.5ul ligation system solution to do transformation.

Go Gel Electrophoresis and Gel Extraction of ⑧, ⑩, 11

Time: 15:30

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

SUSTech Shenzhen-FF23D23B-B68F-42EB-87A2-DBDED38BDF8E.png

Gel extraction concentration: ⑩: 67.3 ng/ul 1.65; 11: 66.4 ng/ul 1.69.

‘’‘Note:’‘’ We found that we take a wrong ⑧ before.

Transformation of ⑧

Time: 16:30

Handler: Tang Shiqiang

Procedure: IGEM Transformation Protocol

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑨+⑤

Time: 19:30

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
Incubate overnight at 37℃, 220rpm.

8.1

Picking single colonies of ⑧

Time: 9:30

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
Incubate at 37℃ for about 14h, 220rpm.

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B

Time: 9:30

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑥+⑤A: 126.3 ng/ul 1.86; ⑥+⑤B: 124.9 ng/ul 1.82; ⑦+⑤A: 116.5 ng/ul 1.87; ⑦+⑤B: 112.0 ng/ul 1.86; ⑨+⑤A: 120.4 ng/ul 1.87; ⑨+⑤B: 149.0 ng/ul 1.87;

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B

Time: 12:00

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑥+⑤A	⑦+⑤A	⑨+⑤A	⑥+⑤B	⑦+⑤B	⑨+⑤B
DNA(200ng)	2ul	2ul	2ul	2ul	2ul	2ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul
EcoRI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
PstI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	15.6ul	15.6ul	15.6ul	15.6ul	15.6ul	15.6ul
Total	20ul	20ul	20ul	20ul	20ul	20ul

Incubate at 37℃ for 30 min.
Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.

Results:

SUSTech Shenzhen-3DD57F02-827C-43BC-B57F-2ACCA230ECF4.png

‘’‘Comments:’‘’ The length of them are incorrect. Their length are larger than 1000bp while should be smaller than 1000bp.

‘’‘Note:’‘’ Finally we found that we used a tube of wrong ⑤.

Put ⑧ A and B at 4℃

Time: 20:00

Handler: Tang Shiqiang

8.2

Plasmid extraction of ⑧ A and B

Time: 14:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑧A: 116.5 ng/ul 1.82; ⑧B: 73.2 ng/ul 1.81.

Enzyme Digestion for Gel Extraction of ⑤, ⑧

Time: 22:00

Handler: Lu Shixin, Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑤3-3-F 1
DNA（>3ug）	21.5ul
Cutsmart buffer	2.5ul
EcoRI-HF	0.5ul
XbaI	0.5ul
ddH₂O	0ul
Total	25ul

	⑧A
DNA (>2ug)	21.5ul
Cutsmart buffer	2.5ul
EcoRI-HF	0.5ul
SpeI-HF	0.5ul
ddH₂O	0ul
Total	25ul

Incubate at 37℃ overnight. (Start from 22:25)

8.3

Go Gel Electrophoresis and Gel Extraction of ⑤, ⑧

Time: 10:00

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

SUSTech Shenzhen-0D26F48D-11BF-4C72-B460-51AB3110125F.png

Concentration: ⑤: 51.4 ng/ul 1.75; ⑧: 23.2 ng/ul 1.71.

Ligation of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 16:30

Handler: Lu Shixin

Procedure:

Make ligation system solution.

	⑥+⑤	⑦+⑤	⑧+⑤	⑨+⑤
Vector (50ng)	1ul	1ul	1ul	1ul
Insert	1.5ul	1.5ul	2.5ul	2ul
ddH₂O	7.5ul	7.5ul	6.5ul	7ul
2x Quick ligase buffer	10ul	10ul	10ul	10ul
Quick ligase	1ul	1ul	1ul	1ul
Total	21ul	21ul	21ul	21ul

Incubate at room temperature for 10 min.
Take 2ul ligation system solution to do transformation.

8.4

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 9:30

Handler: Lu Shixin

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
Incubate at 37℃ for about 12h, 220rpm.

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B

Time: 21:30

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑥+⑤A: 54.4 ng/ul 2.18; ⑥+⑤B: 29.2 ng/ul 3.15; ⑦+⑤A: 55.4 ng/ul 2.42; ⑦+⑤B: 58.8 ng/ul 2.42; ⑧+⑤A: 70.0 ng/ul 2.22; ⑧+⑤B: 35.6 ng/ul 2.84; ⑨+⑤A: 65.9 ng/ul 2.23; ⑨+⑤B: 74.3 ng/ul 2.18;

‘’’Comments:’’’ All the concentration seems to have some problem, and the A260/280 have some problem.

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B

Time: 23:00

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑥+⑤A	⑦+⑤A	⑧+⑤A	⑨+⑤A	⑥+⑤B	⑦+⑤B	⑧+⑤B	⑨+⑤B
DNA(200ng)	4ul	4ul	3ul	3ul	7ul	4ul	7ul	3ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul	2ul	2ul
XbaI	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
PstI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	13.6ul	13.6ul	14.6ul	14.6ul	10.6ul	13.6ul	10.6ul	14.6ul
Total	20ul	20ul	20ul	20ul	20ul	20ul	20ul	20ul

Incubate at 37℃ for 30 min.
Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.

Results:

The length seems to be correct.

8.5

Enzyme Digestion for Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 2:20

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑥+⑤A	⑦+⑤B	⑧+⑤A	⑨+⑤B
DNA（>1.5ug）	26ul	26ul	26ul	26ul
Cutsmart buffer	3ul	3ul	3ul	3ul
EcoRI-HF	0.5ul	0.5ul	0.5ul	0.5ul
XbaI	0.5ul	0.5ul	0.5ul	0.5ul
ddH₂O	0ul	0ul	0ul	0ul
Total	30ul	30ul	30ul	30ul

Incubate at 37℃ overnight. (Start from 2:30)

Go Gel Electrophoresis and Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 8:10

Handler: Lu Shixin, Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

Concentration: ⑥+⑤: 25.1 ng/ul 1.58; ⑦+⑤: 23.4 ng/ul 1.54; ⑧+⑤: 45.9 ng/ul 1.01; ⑨+⑤: 26.7 ng/ul 1.50;

Ligation of ⑩,11 and ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 11:00

Handler: Lu Shixin, Tang Shiqiang

Procedure:

Make ligation system solution.

	⑩+⑥+⑤	⑩+⑦+⑤	⑩+⑧+⑤	⑩+⑨+⑤
Vector ⑩ (50ng)	0.8ul	0.8ul	0.8ul	0.8ul
Insert	3.4ul	3.4ul	2ul	3.4ul
ddH₂O	5.8ul	5.8ul	7.2ul	5.8ul
2x Quick ligase buffer	10ul	10ul	10ul	10ul
Quick ligase	1ul	1ul	1ul	1ul
Total	21ul	21ul	21ul	21ul

	11+⑥+⑤	11+⑦+⑤	11+⑧+⑤	11+⑨+⑤
Vector ⑩ (50ng)	0.8ul	0.8ul	0.8ul	0.8ul
Insert	3.4ul	3.4ul	2ul	3.4ul
ddH₂O	5.8ul	5.8ul	7.2ul	5.8ul
2x Quick ligase buffer	10ul	10ul	10ul	10ul
Quick ligase	1ul	1ul	1ul	1ul
Total	21ul	21ul	21ul	21ul

Incubate at room temperature for 10 min.
Take 5ul ligation system solution to do transformation.

8.6

Observation of single colonies of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 7:00

Handler: Xiao Tianyao

Results:

	Colonies number
⑩ backbone control	5
11 backbone control	20
⑩+⑥+⑤	None
⑩+⑦+⑤	1
⑩+⑧+⑤	20
⑩+⑨+⑤	>50
11+⑥+⑤	4
11+⑦+⑤	1
11+⑧+⑤	6
11+⑨+⑤	>50

Comments: Control groups have colonies. So the ligation results are improbable. We would redo it.

Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 10:00

Handler: Liao Weiduo

Procedure:

Pick single colonies, adding 3 ml C+ LB.
Incubate at 37℃ for about 12h, 220rpm.

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 11:00

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, C and D.
Incubate at 37℃ for about 12h, 220rpm.

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B

Time: 23:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑥+⑤C: 63.8 ng/ul 1.81; ⑥+⑤D: 59.4 ng/ul 1.79; ⑦+⑤C: 49.9 ng/ul 1.81; ⑦+⑤D: 65.8 ng/ul 1.75; ⑧+⑤C: 73.4ng/ul 1.81; ⑧+⑤D: 72.8 ng/ul 1.81; ⑨+⑤C: 65.8 ng/ul 1.74; ⑨+⑤D: 64.1 ng/ul 1.83.

8.7

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ C and D

Time: 2:00

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑥+⑤C	⑦+⑤C	⑧+⑤C	⑨+⑤C	⑥+⑤D	⑦+⑤D	⑧+⑤D	⑨+⑤D
DNA(100ng)	1.5ul	1.5ul	1.5ul	1.5ul	1.5ul	1.5ul	1.5ul	1.5ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul	2ul	2ul
XbaI	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
PstI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	16.1ul	16.1ul	16.1ul	16.1ul	16.1ul	16.1ul	16.1ul	1.16ul
Total	20ul	20ul	20ul	20ul	20ul	20ul	20ul	20ul

Incubate at 37℃ for 30 min.
Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.

Results:

SUSTech Shenzhen-1C4A2A62-E9CB-49BE-B41A-544A82C84E24.png

Observation of LB medium color of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 7:00

Handler: Xiao Tianyao

Results:

	Color
⑩+⑥+⑤	None
⑩+⑦+⑤	Purple
⑩+⑧+⑤	Yellow
⑩+⑨+⑤	Red
11+⑥+⑤	Red
11+⑦+⑤	None
11+⑧+⑤	None
11+⑨+⑤	None

‘’’Note:’’’ There are yellow colonies on plate 11+⑧+⑤ and red colonies on 11+⑨+⑤.

Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤ （Because there are obvious correct color colonies on those plates）

Time: 9:30

Handler: Liao Weiduo

Procedure:

Pick single colonies, adding 6 ml C+ LB.
Incubate at 37℃ for about 12h, 220rpm.

Ligation of ⑩ and ⑥+⑤, 11 and ⑦+⑤

Time: 11:00

Handler: Tang Shiqiang

Procedure:

Make ligation system solution.

	⑩+⑥+⑤
Vector ⑩ (50ng)	0.7ul
Insert	3.2ul
ddH₂O	6.1ul
2x Quick ligase buffer	10ul
Quick ligase	1ul
Total	21ul

	11+⑦+⑤
Vector ⑩ (50ng)	0.7ul
Insert	3.2ul
ddH₂O	6.1ul
2x Quick ligase buffer	10ul
Quick ligase	1ul
Total	21ul

Incubate at room temperature for 10 min.
Take 5ul ligation system solution to do transformation.

Plasmid extraction of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 22:00

Handler: Liao Weiduo

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑩+⑦+⑤: 63.6 ng/ul 1.71; ⑩+⑧+⑤: 100.7 ng/ul 1.54; ⑩+⑨+⑤: 83.0 ng/ul 1.59; 11+⑥+⑤: 114.8 ng/ul 1.65; 11+⑧+⑤: 77.5 ng/ul 1.78; 11+⑨+⑤: 106.3 ng/ul 1.51.

8.9

Picking single colonies of ⑩+⑥+⑤, 11+⑦+⑤

Time: 00:00

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics.
Incubate overnight at 37℃, 220rpm.

Plasmid extraction of ⑩+⑥+⑤, 11+⑦+⑤

Time: 16:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑩+⑥+⑤: 109.0 ng/ul 1.74; 11+⑦+⑤: 115.3 ng/ul 1.85.

sfGFP notebook

8.2

Plate streaking of a plasmid with contain sfGFP and msfGFP

Time: 17:30

Handler: Tang Shiqiang

8.3

Picking single colonies of sfGFP and msfGFP

Time: 9:00

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics.
Incubate for about 12h at 37℃, 220rpm.

Plasmid extraction of sfGFP and msfGFP

Time: 21:30

Handler: Lu Shixin

Procedure:bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: sfGFP: 70.0 ng/ul 1.77; msfGFP: 460.8 ng/ul.

8.16

PCR of sfGFP and msfGFP

Time: 16:00

Handler: Tang Shiqiang

Procedure:

DNA	0.1ul
bbp3	2.5ul
bbp4	2.5ul
Q5	25ul
ddH₂O	20ul
Total	50ul

32 cycles	Temperature	Time
Initialization	98℃	30s
Denaturation	98℃	5s
Annealing	65℃	20s
Extension	72℃	10s
Final elongation	72℃	2min

Go Gel Electrophoresis and Gel Extraction of sfGFP and msfGFP PCR product

Time: 19:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Result:

Concentration: sfGFP: 43.2 ng/ul 1.76; msfGFP: 62.0 ng/ul 1.79.

Enzyme Digestion for Gel Extraction of sfGFP and msfGFP

Time: 19:30

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	sfGFP	msfGFP
DNA（>1ug）	27.5ul	27.5ul
Cutsmart buffer	2.5ul	2.5ul
EcoRI-HF	0.5ul	0.5ul
SpeI-HF	0.5ul	0.5ul
ddH₂O	19ul	19ul
Total	50ul	50ul

Incubate at 37℃ for 3h.

Enzyme Digestion for Gel Extraction of ⑤

Time: 17:00

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	⑤
DNA（>3ug）	21.5ul
Cutsmart buffer	2.5ul
EcoRI-HF	0.5ul
SpeI-HF	0.5ul
ddH₂O	0
Total	25ul

Incubate at 37℃ for 3h

Go Gel Electrophoresis and Gel Extraction of sfGFP, msfGFP, and ⑤

Time: 20:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

145x256px

Gel extraction concentration: ⑤: 61.4 ng/ul 1.58; sfGFP: 16.9 ng/ul 1.62; msfGFP: 16.5 ng/ul 1.59.

8.17

Ligation of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3

Time: 0:00

Handler: Tang Shiqiang

Procedure:

Make ligation system solution.

	sfGFP	msfGFP
Vector ⑤ (50ng)	1ul	1ul
Insert	4ul	4ul
ddH₂O	0ul	0ul
2x Quick ligase buffer	5ul	5ul
Quick ligase	0.5ul	0.5ul
Total	10.5ul	10.5ul

Incubate at room temperature for 10 min.
Take 10.5ul ligation system solution to do transformation.

Picking single colonies of sfGFP and msfGFP

Time: 18:30

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 6ml LB with corresponding antibiotics.
Incubate overnight at 37℃, 220rpm.

8.18

Plasmid extraction of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3

Time: 13:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: sfGFP+⑤: 103.6 ng/ul 1.96; msfGFP+⑤: 127.1 ng/ul 1.80; sfGFP+pSB1C3①: 156.1 ng/ul 1.91; sfGFP+pSB1C3②: 179.2 ng/ul 1.91; msfGFP+pSB1C3①: 115.5 ng/ul 1.93; msfGFP+pSB1C3②: 159.5 ng/ul 1.92.

8.18

Enzyme Digestion and Go Gel Electrophoresis Test of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3

Time: 0:00

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	sfGFP+⑤	msfGFP+⑤	sfGFP+pSB1C3①	sfGFP+pSB1C3②	msfGFP+pSB1C3①	msfGFP+pSB1C3②
DNA(200ng)	2ul	2ul	2ul	2ul	2ul	2ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul
KpnI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	16.8ul	16.8ul	16.8ul	16.8ul	16.8ul	16.8ul
Total	20ul	20ul	20ul	20ul	20ul	20ul

	sfGFP+⑤	msfGFP+⑤	sfGFP+pSB1C3①	sfGFP+pSB1C3②	msfGFP+pSB1C3①	msfGFP+pSB1C3②
DNA(200ng)	2ul	2ul	2ul	2ul	2ul	2ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul
EcoRI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	16.8ul	16.8ul	16.8ul	16.8ul	16.8ul	16.8ul
Total	20ul	20ul	20ul	20ul	20ul	20ul

	sfGFP+⑤	msfGFP+⑤	sfGFP+pSB1C3①	sfGFP+pSB1C3②	msfGFP+pSB1C3①	msfGFP+pSB1C3②
DNA(200ng)	2ul	2ul	2ul	2ul	2ul	2ul
Cutsmart buffer	2ul	2ul	2ul	2ul	2ul	2ul
EcoRI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
PstI-HF	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul	0.2ul
ddH₂O	16.6ul	16.6ul	16.6ul	16.6ul	16.6ul	16.6ul
Total	20ul	20ul	20ul	20ul	20ul	20ul

Incubate overnight at 37℃.
Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.

Results:

553x273px

9.21

PCR of msfGFP Coding sequence basic part

Time: 16:00

Handler: Tang Shiqiang

Procedure:

DNA	1ul
SfGFP-forward	1ul
bbp4	1ul
primeSTAR	20ul
ddH₂O	17ul
Total	40ul

32 cycles	Temperature	Time
Initialization	98℃	30s
Denaturation	98℃	10s
Annealing	65℃	5s
Extension	72℃	5s
Final elongation	72℃	2min

Go Gel Electrophoresis and Gel Extraction of msfGFP CDS

Time: 20:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Result:

Concentration: msfGFP: 96.1 ng/ul 1.83.

Enzyme Digestion for Gel Extraction of msfGFP CDS

Time: 20:30

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	msfGFP CDS
DNA	43ul
Cutsmart buffer	5ul
EcoRI-HF	1ul
SpeI-HF	1ul
ddH₂O	0ul
Total	50ul

Incubate overnight at 37℃.

9.22

Transformation of msfGFP CDS

Time: 11:00

Handler: Tang Shiqiang

Procedure: IGEM Protocols

Picking single colonies of sfGFP CDS

Time: 23:00

Handler: Tang Shiqiang

Procedure:

Pick single colonies, adding 5ml C+ LB.
Incubate overnight at 37℃, 220rpm.

9.23

Plasmid extraction of msfGFP CDS

Time: 17:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: msfGFP CDS①: 141.0 ng/ul 1.88; msfGFP CDS②: 141.0 ng/ul 1.90.

9.24

Enzyme Digestion and Go Gel Electrophoresis Test of msfGFP CDS① and msfGFP CDS②

Time: 11:40

Handler: Tang Shiqiang

Procedure:

Make enzyme cutting system solution.

	msfGFP CDS①	msfGFP CDS②
DNA(300ng)	2.5ul	2.5ul
Cutsmart buffer	1ul	1ul
MluI-HF/MscI	0.5ul	0.5ul
ddH₂O	6ul	6ul
Total	10ul	10ul

Incubate for 3h at 37℃.
Make 1% agarose gel 30ml.
Add 6x loading dye to the enzyme cutting system solution.
Go gel electrophoresis.

Results:

389x357px

@@ Line 6: / Line 6: @@
 {{:Team:SUSTech_Shenzhen/main-content-begin}}
-== 8 Chromoprotein ==
+= 8 Chromoprotein =
-=== Plasmid Construction ===
+== Plasmid Construction ==
 ALL ABBREVIATIONS USED:
@@ Line 98: / Line 98: @@
 | 57
 |}
-=== 7.22 ===
+== 7.22 ==
-==== Transformation of ①, ③, ⑤ ====
+=== Transformation of ①, ③, ⑤ ===
 '''Time:''' 20:30
@@ Line 105: / Line 105: @@
 '''Handler:''' Liao Weiduo
-===== Procedure: IGEM Protocols =====
+==== Procedure: IGEM Protocols ====
 . Thaw competent cells on ice.
@@ Line 131: / Line 131: @@
 . Incubate transformations overnight at 37℃.
-=== 7.23 ===
+== 7.23 ==
-==== Picking single colonies of ①, ③, ⑤ ====
+=== Picking single colonies of ①, ③, ⑤ ===
 '''Time:''' 16:00
@@ Line 138: / Line 138: @@
 '''Handler:''' Liao Weiduo
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
 # Incubate overnight at 37℃, 220rpm.
-=== 7.24 ===
+== 7.24 ==
-==== Plasmid extraction of ①, ③, ⑤ A and B ====
+=== Plasmid extraction of ①, ③, ⑤ A and B ===
 '''Time:''' 9:00
@@ Line 149: / Line 149: @@
 '''Handler:''' Liao Weiduo
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
 # Add 2ml LB medium to a 2ml microcentrifuge tube.
@@ Line 176: / Line 176: @@
 # Centrifuge at maximum speed for 1 min.
-===== Results: =====
+==== Results: ====
 ‘’’Concentration:’’’ ①A: 245.9 ng/ul 1.84; ①B: 340.5 ng/ul 1.84; ③A: 116.2 ng/ul 1.65; ③B: 102.1 ng/ul 1.77; ⑤A: 151.4 ng/ul 1.80; ⑤B: 68.0 ng/ul 1.84;
-=== 7.25 ===
+== 7.25 ==
-==== Transformation of ②,④,⑥,⑦,⑧,⑨ ====
+=== Transformation of ②,④,⑥,⑦,⑧,⑨ ===
 '''Time:''' 0:00
@@ Line 187: / Line 187: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: IGEM Protocols =====
+==== Procedure: IGEM Protocols ====
-==== Picking single colonies of ②,④,⑥,⑦,⑧,⑨  ====
+=== Picking single colonies of ②,④,⑥,⑦,⑧,⑨  ===
 '''Time:''' 20:00
@@ Line 195: / Line 195: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, α and β.
 # Incubate overnight at 37℃, 220rpm.
-=== 7.26 ===
+== 7.26 ==
-==== Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group α ====
+=== Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group α ===
 '''Time:''' 9:40
@@ Line 208: / Line 208: @@
 '''Handler:''' Lu Shixin
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ②α: 369.8 ng/ul; ④α: 142.4 ng/ul; ⑥α: 116.5 ng/ul; ⑦α: 92 ng/ul; ⑧α: 157.4 ng/ul; ⑨α: 168.3 ng/ul.
@@ Line 216: / Line 216: @@
 ‘’’ Note: ’’’ ⑧ has a red colony.
-==== Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group β ====
+=== Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group β ===
 '''Time:''' 14:00
@@ Line 222: / Line 222: @@
 '''Handler:''' Tang Shiqiang
-=====Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+====Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ②β: 496.9 ng/ul 1.86; ④β: 256.4 ng/ul 1.85; ⑥β: 220.3 ng/ul 1.85; ⑦β: 225.7 ng/ul 1.85; ⑧β: 304.6 ng/ul 1.85; ⑨β: 352.2 ng/ul 1.84.
@@ Line 230: / Line 230: @@
 ‘’’ Note: ‘’’ ⑧ has a red colony.
-=== 7.27 ===
+== 7.27 ==
-==== Competent Cell Test ====
+=== Competent Cell Test ===
 '''Time:''' 16:00
@@ Line 238: / Line 238: @@
 '''Handler:''' Lu Shixin
-===== Procedure: IGEM Protocols =====
+==== Procedure: IGEM Protocols ====
 # Spin down the DNA tubes form the Competent Cell Test Kit to collect all of the DNA into the bottom of each tube prior to use.
@@ Line 251: / Line 251: @@
 # Incubate at 37℃ overnight, approximately 16 h.
-==== Enzyme Digestion and Go Gel Electrophoresis Test of ②,④,⑥,⑦,⑧,⑨ ====
+=== Enzyme Digestion and Go Gel Electrophoresis Test of ②,④,⑥,⑦,⑧,⑨ ===
 '''Time:''' 20:00
@@ Line 257: / Line 257: @@
 '''Handler:''' Xiao Tianyao
-===== Procedure: =====
+==== Procedure: ====
 . Make enzyme cutting system solution.
@@ Line 400: / Line 400: @@
 <li><p>Go gel electrophoresis.</p></li></ol>
-===== Results: =====
+==== Results: ====
 figures show below:
@@ Line 406: / Line 406: @@
 {{SUSTech_Image | filename=SUSTech_Shenzhen-9BCCBDAD-C55F-46B0-B96C-D880F0C7F660.png | caption=‘’’Comment:’’’ ⑧ has some problem, and we will redo it. | width=800px}}
-=== 7.28 ===
+== 7.28 ==
-==== Competent Cell Test Colonies Counting ====
+=== Competent Cell Test Colonies Counting ===
 '''Time:''' 13:00
@@ Line 414: / Line 414: @@
 '''Handler:''' Lu Shixin
-===== Result: =====
+==== Result: ====
 {{SUSTech_Image | filename=SUSTech_Shenzhen-C89646A9-AD79-4481-95BB-E54BBC7C1B87.png | caption=Figure 1. Water control | width=300px}}
 {{SUSTech_Image | filename=SUSTech_Shenzhen-D6D86280-E0BE-43CE-A096-8C835F4D8426.png | caption= Figure 2. 0.5 pg/ul | width=300px}}
@@ Line 422: / Line 422: @@
 And all the colonies are red under UV light.
-=== 7.29 ===
+== 7.29 ==
-==== Transformation of ⑧,⑩，11 ====
+=== Transformation of ⑧,⑩，11 ===
 '''Time:''' 16:00
@@ Line 430: / Line 430: @@
 '''Handler:''' Lu Shixin, Tang Shiqiang
-===== Procedure: IGEM Transformation Protocol =====
+==== Procedure: IGEM Transformation Protocol ====
-==== Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑧,⑨ ====
+=== Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑧,⑨ ===
 '''Time:''' 20:00
@@ Line 438: / Line 438: @@
 '''Handler:''' LuShixin, Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 519: / Line 519: @@
 # Incubate at 37℃ overnight. (Start from 21:20)
-=== 7.30 ===
+== 7.30 ==
-==== Picking single colonies of ⑧, ⑩, 11 ====
+=== Picking single colonies of ⑧, ⑩, 11 ===
 '''Time:''' 9:00
@@ Line 527: / Line 527: @@
 '''Handler:''' Lu Shixin
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 1ml LB with corresponding antibiotics in 1.5ml EP tube, each plate we pick 2 single colonies, α and β.
@@ Line 534: / Line 534: @@
 # Incubate at 37℃ for 10h, 220rpm.
-==== Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑧,⑨ ====
+=== Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑧,⑨ ===
 '''Time:''' 9:00
@@ Line 540: / Line 540: @@
 '''Handler:''' Lu Shixin
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
 # Make 1% agarose gel 30ml.
@@ Line 567: / Line 567: @@
 # Centrifuge at maximum speed for 1 min.
-===== Results： =====
+==== Results： ====
 {{SUSTech_Image | filename=SUSTech_Shenzhen-4E6C74F1-3B4C-4F1A-B283-6751068590D8.png | caption= | width=500px}}
@@ Line 573: / Line 573: @@
 All the concentration of gel extraction are below 10 ng/ul. So we decide to repeat enzyme digestion with more DNA.
-==== Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑨ (Redo) ====
+=== Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑨ (Redo) ===
 '''Time:''' 18:00
@@ Line 579: / Line 579: @@
 '''Handler:''' Lu Shixin
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 647: / Line 647: @@
 # Incubate at 37℃ for 3h. (Start from 18:40)
-==== Plasmid extraction of ⑧,⑩,11 ====
+=== Plasmid extraction of ⑧,⑩,11 ===
 '''Time:''' 21:00
@@ Line 653: / Line 653: @@
 '''Handler:''' Lu Shixin
-===== Procedure:''' '''bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure:''' '''bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 The length of them are correct.
@@ Line 661: / Line 661: @@
 Concentration: ⑧: 130.2 ng/ul; ⑩: 147.0 ng/ul; 11: 93.3 ng/ul.
-==== Enzyme Digestion for Gel Extraction of ⑧,⑩,11 ====
+=== Enzyme Digestion for Gel Extraction of ⑧,⑩,11 ===
 '''Time:''' 22:30
@@ Line 667: / Line 667: @@
 '''Handler:''' Lu Shixin
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 726: / Line 726: @@
 # Incubate at 37℃ overnight. (Start from 22:40)
-==== Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑨ (Redo) ====
+=== Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑨ (Redo) ===
 '''Time:''' 22:00
@@ Line 732: / Line 732: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Gel extraction concentration: ⑤: 15.3 ng/ul; ⑥: 36.3 ng/ul; ⑦: 36.8 ng/ul; ⑨: 26.9 ng/ul.
-=== 7.31 ===
+== 7.31 ==
-==== Ligation of ⑥+⑤, ⑦+⑤, ⑨+⑤ ====
+=== Ligation of ⑥+⑤, ⑦+⑤, ⑨+⑤ ===
 '''Time:''' 0:00
@@ Line 746: / Line 746: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make ligation system solution.
@@ Line 790: / Line 790: @@
 # Take 2.5ul ligation system solution to do transformation.
-==== Go Gel Electrophoresis and Gel Extraction of ⑧, ⑩, 11 ====
+=== Go Gel Electrophoresis and Gel Extraction of ⑧, ⑩, 11 ===
 '''Time:''' 15:30
@@ Line 796: / Line 796: @@
 '''Handler:''' Lu Shixin
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Results: =====
+==== Results: ====
 {{SUSTech_Image | filename=SUSTech_Shenzhen-FF23D23B-B68F-42EB-87A2-DBDED38BDF8E.png | caption= | width=500px}}
@@ Line 806: / Line 806: @@
 ‘’‘Note:’‘’ We found that we take a wrong ⑧ before.
-==== Transformation of ⑧ ====
+=== Transformation of ⑧ ===
 '''Time:''' 16:30
@@ Line 812: / Line 812: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: IGEM Transformation Protocol =====
+==== Procedure: IGEM Transformation Protocol ====
-==== Picking single colonies of ⑥+⑤, ⑦+⑤, ⑨+⑤ ====
+=== Picking single colonies of ⑥+⑤, ⑦+⑤, ⑨+⑤ ===
 '''Time:''' 19:30
@@ Line 820: / Line 820: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
 # Incubate overnight at 37℃, 220rpm.
-=== 8.1 ===
+== 8.1 ==
-==== Picking single colonies of ⑧ ====
+=== Picking single colonies of ⑧ ===
 '''Time:''' 9:30
@@ Line 833: / Line 833: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
 # Incubate at 37℃ for about 14h, 220rpm.
-==== Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B ====
+=== Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B ===
 '''Time:''' 9:30
@@ Line 844: / Line 844: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑥+⑤A: 126.3 ng/ul 1.86; ⑥+⑤B: 124.9 ng/ul 1.82; ⑦+⑤A: 116.5 ng/ul 1.87; ⑦+⑤B: 112.0 ng/ul 1.86; ⑨+⑤A: 120.4 ng/ul 1.87; ⑨+⑤B: 149.0 ng/ul 1.87;
-==== Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B ====
+=== Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B ===
 '''Time:''' 12:00
@@ Line 856: / Line 856: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 924: / Line 924: @@
 # Go gel electrophoresis.
-===== Results: =====
+==== Results: ====
 {{SUSTech_Image | filename=SUSTech_Shenzhen-3DD57F02-827C-43BC-B57F-2ACCA230ECF4.png | caption= | width=500px}}
@@ Line 931: / Line 931: @@
 ‘’‘Note:’‘’ Finally we found that we used a tube of wrong ⑤.
-==== Put ⑧ A and B at 4℃ ====
+=== Put ⑧ A and B at 4℃ ===
 '''Time:''' 20:00
@@ Line 937: / Line 937: @@
 '''Handler:''' Tang Shiqiang
-=== 8.2 ===
+== 8.2 ==
-==== Plasmid extraction of ⑧ A and B ====
+=== Plasmid extraction of ⑧ A and B ===
 '''Time:''' 14:00
@@ Line 945: / Line 945: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑧A: 116.5 ng/ul 1.82; ⑧B: 73.2 ng/ul 1.81.
-==== Enzyme Digestion for Gel Extraction of ⑤, ⑧ ====
+=== Enzyme Digestion for Gel Extraction of ⑤, ⑧ ===
 '''Time:''' 22:00
@@ Line 957: / Line 957: @@
 '''Handler:''' Lu Shixin, Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 1,011: / Line 1,011: @@
 # Incubate at 37℃ overnight. (Start from 22:25)
-=== 8.3 ===
+== 8.3 ==
-==== Go Gel Electrophoresis and Gel Extraction of ⑤, ⑧ ====
+=== Go Gel Electrophoresis and Gel Extraction of ⑤, ⑧ ===
 '''Time:''' 10:00
@@ Line 1,019: / Line 1,019: @@
 '''Handler:''' Lu Shixin
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Results: =====
+==== Results: ====
 {{SUSTech_Image | filename=SUSTech_Shenzhen-0D26F48D-11BF-4C72-B460-51AB3110125F.png | caption= | width=500px}}
 Concentration: ⑤: 51.4 ng/ul 1.75; ⑧: 23.2 ng/ul 1.71.
-==== Ligation of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ====
+=== Ligation of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ===
 '''Time:''' 16:30
@@ Line 1,032: / Line 1,032: @@
 '''Handler:''' Lu Shixin
-===== Procedure: =====
+==== Procedure: ====
 # Make ligation system solution.
@@ Line 1,084: / Line 1,084: @@
 # Take 2ul ligation system solution to do transformation.
-=== 8.4 ===
+== 8.4 ==
-==== Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ====
+=== Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ===
 '''Time:''' 9:30
@@ Line 1,092: / Line 1,092: @@
 '''Handler:''' Lu Shixin
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
 # Incubate at 37℃ for about 12h, 220rpm.
-==== Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B ====
+=== Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B ===
 '''Time:''' 21:30
@@ Line 1,103: / Line 1,103: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑥+⑤A: 54.4 ng/ul 2.18; ⑥+⑤B: 29.2 ng/ul 3.15; ⑦+⑤A: 55.4 ng/ul 2.42; ⑦+⑤B: 58.8 ng/ul 2.42; ⑧+⑤A: 70.0 ng/ul 2.22; ⑧+⑤B: 35.6 ng/ul 2.84; ⑨+⑤A: 65.9 ng/ul 2.23; ⑨+⑤B: 74.3 ng/ul 2.18;
@@ Line 1,111: / Line 1,111: @@
 ‘’’Comments:’’’ All the concentration seems to have some problem, and the A260/280 have some problem.
-==== Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B ====
+=== Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B ===
 '''Time:''' 23:00
@@ Line 1,117: / Line 1,117: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 1,199: / Line 1,199: @@
 # Go gel electrophoresis.
-===== Results: =====
+==== Results: ====
 The length seems to be correct.
-=== 8.5 ===
+== 8.5 ==
-==== Enzyme Digestion for Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ====
+=== Enzyme Digestion for Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ===
 '''Time:''' 2:20
@@ Line 1,211: / Line 1,211: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 1,263: / Line 1,263: @@
 <li><p>Incubate at 37℃ overnight. (Start from 2:30)</p></li></ol>
-==== Go Gel Electrophoresis and Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ====
+=== Go Gel Electrophoresis and Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ===
 '''Time:''' 8:10
@@ Line 1,269: / Line 1,269: @@
 '''Handler:''' Lu Shixin, Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑥+⑤: 25.1 ng/ul 1.58; ⑦+⑤: 23.4 ng/ul 1.54; ⑧+⑤: 45.9 ng/ul 1.01; ⑨+⑤: 26.7 ng/ul 1.50;
-==== Ligation of ⑩,11 and ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ====
+=== Ligation of ⑩,11 and ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ===
 '''Time:''' 11:00
@@ Line 1,281: / Line 1,281: @@
 '''Handler:''' Lu Shixin, Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make ligation system solution.
@@ Line 1,378: / Line 1,378: @@
 # Take 5ul ligation system solution to do transformation.
-=== 8.6 ===
+== 8.6 ==
-==== Observation of single colonies of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤ ====
+=== Observation of single colonies of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤ ===
 '''Time:''' 7:00
@@ Line 1,386: / Line 1,386: @@
 '''Handler:''' Xiao Tianyao
-===== Results: =====
+==== Results: ====
 {| class=" table table-striped"
@@ Line 1,425: / Line 1,425: @@
 Comments: Control groups have colonies. So the ligation results are improbable. We would redo it.
-==== Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤ ====
+=== Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤ ===
 '''Time:''' 10:00
@@ Line 1,431: / Line 1,431: @@
 '''Handler:''' Liao Weiduo
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 3 ml C+ LB.
 # Incubate at 37℃ for about 12h, 220rpm.
-==== Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ====
+=== Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ ===
 '''Time:''' 11:00
@@ Line 1,442: / Line 1,442: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, C and D.
 # Incubate at 37℃ for about 12h, 220rpm.
-==== Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B ====
+=== Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B ===
 '''Time:''' 23:00
@@ Line 1,453: / Line 1,453: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑥+⑤C: 63.8 ng/ul 1.81; ⑥+⑤D: 59.4 ng/ul 1.79; ⑦+⑤C: 49.9 ng/ul 1.81; ⑦+⑤D: 65.8 ng/ul 1.75; ⑧+⑤C: 73.4ng/ul 1.81; ⑧+⑤D: 72.8 ng/ul 1.81; ⑨+⑤C: 65.8 ng/ul 1.74; ⑨+⑤D: 64.1 ng/ul 1.83.
-=== 8.7 ===
+== 8.7 ==
-==== Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ C and D ====
+=== Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ C and D ===
 '''Time:''' 2:00
@@ Line 1,467: / Line 1,467: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 1,549: / Line 1,549: @@
 # Go gel electrophoresis.
-===== Results: =====
+==== Results: ====
 {{SUSTech_Image | filename=SUSTech_Shenzhen-1C4A2A62-E9CB-49BE-B41A-544A82C84E24.png | caption= | width=500px}}
-==== Observation of LB medium color of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤ ====
+=== Observation of LB medium color of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤ ===
 '''Time:''' 7:00
@@ Line 1,558: / Line 1,558: @@
 '''Handler:''' Xiao Tianyao
-===== Results: =====
+==== Results: ====
 {|  class=" table table-striped"
@@ Line 1,592: / Line 1,592: @@
 ‘’’Note:’’’ There are yellow colonies on plate 11+⑧+⑤ and red colonies on 11+⑨+⑤.
-==== Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤ （Because there are obvious correct color colonies on those plates）====
+=== Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤ （Because there are obvious correct color colonies on those plates）===
 '''Time:''' 9:30
@@ Line 1,598: / Line 1,598: @@
 '''Handler:''' Liao Weiduo
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6 ml C+ LB.
 # Incubate at 37℃ for about 12h, 220rpm.
-==== Ligation of ⑩ and ⑥+⑤, 11 and ⑦+⑤ ====
+=== Ligation of ⑩ and ⑥+⑤, 11 and ⑦+⑤ ===
 '''Time:''' 11:00
@@ Line 1,609: / Line 1,609: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make ligation system solution.
@@ Line 1,664: / Line 1,664: @@
 # Take 5ul ligation system solution to do transformation.
-==== Plasmid extraction of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤ ====
+=== Plasmid extraction of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤ ===
 '''Time:''' 22:00
@@ Line 1,670: / Line 1,670: @@
 '''Handler:''' Liao Weiduo
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑩+⑦+⑤: 63.6 ng/ul 1.71; ⑩+⑧+⑤: 100.7 ng/ul 1.54; ⑩+⑨+⑤: 83.0 ng/ul 1.59; 11+⑥+⑤: 114.8 ng/ul 1.65; 11+⑧+⑤: 77.5 ng/ul 1.78; 11+⑨+⑤: 106.3 ng/ul 1.51.
-=== 8.9 ===
+== 8.9 ==
-==== Picking single colonies of ⑩+⑥+⑤, 11+⑦+⑤ ====
+=== Picking single colonies of ⑩+⑥+⑤, 11+⑦+⑤ ===
 '''Time:''' 00:00
@@ Line 1,684: / Line 1,684: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics.
 # Incubate overnight at 37℃, 220rpm.
-===== Plasmid extraction of ⑩+⑥+⑤, 11+⑦+⑤ =====
+==== Plasmid extraction of ⑩+⑥+⑤, 11+⑦+⑤ ====
 '''Time:''' 16:00
@@ Line 1,695: / Line 1,695: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Results: =====
+==== Results: ====
 Concentration: ⑩+⑥+⑤: 109.0 ng/ul 1.74; 11+⑦+⑤: 115.3 ng/ul 1.85.
-== sfGFP notebook ==
+= sfGFP notebook =
-=== 8.2 ===
+== 8.2 ==
-==== Plate streaking of a plasmid with contain sfGFP and msfGFP ====
+=== Plate streaking of a plasmid with contain sfGFP and msfGFP ===
 '''Time:''' 17:30
@@ Line 1,711: / Line 1,711: @@
 '''Handler:''' Tang Shiqiang
-=== 8.3 ===
+== 8.3 ==
-==== Picking single colonies of sfGFP and msfGFP ====
+=== Picking single colonies of sfGFP and msfGFP ===
 '''Time:''' 9:00
@@ Line 1,719: / Line 1,719: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics.
 # Incubate for about 12h at 37℃, 220rpm.
-==== Plasmid extraction of sfGFP and msfGFP ====
+=== Plasmid extraction of sfGFP and msfGFP ===
 '''Time:''' 21:30
@@ Line 1,730: / Line 1,730: @@
 '''Handler:''' Lu Shixin
-===== Procedure:bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure:bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Result: =====
+==== Result: ====
 Concentration: sfGFP: 70.0 ng/ul 1.77; msfGFP: 460.8 ng/ul.
-=== 8.16 ===
+== 8.16 ==
-==== PCR of sfGFP and msfGFP ====
+=== PCR of sfGFP and msfGFP ===
 '''Time:''' 16:00
@@ Line 1,744: / Line 1,744: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 {|class="table table-striped"
@@ Line 1,792: / Line 1,792: @@
 |}
-==== Go Gel Electrophoresis and Gel Extraction of sfGFP and msfGFP PCR product ====
+=== Go Gel Electrophoresis and Gel Extraction of sfGFP and msfGFP PCR product ===
 '''Time:''' 19:00
@@ Line 1,798: / Line 1,798: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Result: =====
+==== Result: ====
 Concentration: sfGFP: 43.2 ng/ul 1.76; msfGFP: 62.0 ng/ul 1.79.
-==== Enzyme Digestion for Gel Extraction of sfGFP and msfGFP ====
+=== Enzyme Digestion for Gel Extraction of sfGFP and msfGFP ===
 '''Time:''' 19:30
@@ Line 1,810: / Line 1,810: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 1,846: / Line 1,846: @@
 # Incubate at 37℃ for 3h.
-==== Enzyme Digestion for Gel Extraction of ⑤ ====
+=== Enzyme Digestion for Gel Extraction of ⑤ ===
 '''Time:''' 17:00
@@ Line 1,852: / Line 1,852: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 1,882: / Line 1,882: @@
 # Incubate at 37℃ for 3h
-==== Go Gel Electrophoresis and Gel Extraction of sfGFP, msfGFP, and ⑤ ====
+=== Go Gel Electrophoresis and Gel Extraction of sfGFP, msfGFP, and ⑤ ===
 '''Time:''' 20:00
@@ Line 1,888: / Line 1,888: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Results:=====
+==== Results:====
 [[File:media/image1.png|145x256px]]
@@ Line 1,896: / Line 1,896: @@
 Gel extraction concentration: ⑤: 61.4 ng/ul 1.58; sfGFP: 16.9 ng/ul 1.62; msfGFP: 16.5 ng/ul 1.59.
-=== 8.17===
+== 8.17==
-==== Ligation of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3 ====
+=== Ligation of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3 ===
 '''Time:''' 0:00
@@ Line 1,904: / Line 1,904: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure:=====
+==== Procedure:====
 # Make ligation system solution.
@@ Line 1,942: / Line 1,942: @@
 # Take 10.5ul ligation system solution to do transformation.
-==== Picking single colonies of sfGFP and msfGFP ====
+=== Picking single colonies of sfGFP and msfGFP ===
 '''Time:''' 18:30
@@ Line 1,948: / Line 1,948: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure:=====
+==== Procedure:====
 # Pick single colonies, adding 6ml LB with corresponding antibiotics.
 # Incubate overnight at 37℃, 220rpm.
-=== 8.18 ===
+== 8.18 ==
-==== Plasmid extraction of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3 ====
+=== Plasmid extraction of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3 ===
 '''Time:''' 13:00
@@ Line 1,961: / Line 1,961: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Result:=====
+==== Result:====
 Concentration: sfGFP+⑤: 103.6 ng/ul 1.96; msfGFP+⑤: 127.1 ng/ul 1.80; sfGFP+pSB1C3①: 156.1 ng/ul 1.91; sfGFP+pSB1C3②: 179.2 ng/ul 1.91; msfGFP+pSB1C3①: 115.5 ng/ul 1.93; msfGFP+pSB1C3②: 159.5 ng/ul 1.92.
-=== 8.18 ===
+== 8.18 ==
-==== Enzyme Digestion and Go Gel Electrophoresis Test of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3 ====
+=== Enzyme Digestion and Go Gel Electrophoresis Test of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3 ===
 '''Time:''' 0:00
@@ Line 1,975: / Line 1,975: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 2,145: / Line 2,145: @@
 # Go gel electrophoresis.
-===== Results: =====
+==== Results: ====
 [[File:media/image2.png|553x273px]]
-=== 9.21 ===
+== 9.21 ==
-==== PCR of msfGFP Coding sequence basic part ====
+=== PCR of msfGFP Coding sequence basic part ===
 '''Time:''' 16:00
@@ Line 2,157: / Line 2,157: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 {|class="table table-striped"
@@ Line 2,205: / Line 2,205: @@
 |}
-==== Go Gel Electrophoresis and Gel Extraction of msfGFP CDS ====
+=== Go Gel Electrophoresis and Gel Extraction of msfGFP CDS ===
 '''Time:''' 20:00
@@ Line 2,211: / Line 2,211: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol ====
-===== Result: =====
+==== Result: ====
 Concentration: msfGFP: 96.1 ng/ul 1.83.
-==== Enzyme Digestion for Gel Extraction of msfGFP CDS ====
+=== Enzyme Digestion for Gel Extraction of msfGFP CDS ===
 '''Time:''' 20:30
@@ Line 2,223: / Line 2,223: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 2,253: / Line 2,253: @@
 # Incubate overnight at 37℃.
-=== 9.22 ===
+== 9.22 ==
-==== Transformation of msfGFP CDS ====
+=== Transformation of msfGFP CDS ===
 '''Time:''' 11:00
@@ Line 2,261: / Line 2,261: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: IGEM Protocols =====
+==== Procedure: IGEM Protocols ====
-==== Picking single colonies of sfGFP CDS ====
+=== Picking single colonies of sfGFP CDS ===
 '''Time:''' 23:00
@@ Line 2,269: / Line 2,269: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Pick single colonies, adding 5ml C+ LB.
 # Incubate overnight at 37℃, 220rpm.
-=== 9.23 ===
+== 9.23 ==
-==== Plasmid extraction of msfGFP CDS ====
+=== Plasmid extraction of msfGFP CDS ===
 '''Time:''' 17:00
@@ Line 2,282: / Line 2,282: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol =====
+==== Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol ====
-===== Result: =====
+==== Result: ====
 Concentration: msfGFP CDS①: 141.0 ng/ul 1.88; msfGFP CDS②: 141.0 ng/ul 1.90.
-=== 9.24 ===
+== 9.24 ==
-==== Enzyme Digestion and Go Gel Electrophoresis Test of msfGFP CDS① and msfGFP CDS② ====
+=== Enzyme Digestion and Go Gel Electrophoresis Test of msfGFP CDS① and msfGFP CDS② ===
 '''Time:''' 11:40
@@ Line 2,296: / Line 2,296: @@
 '''Handler:''' Tang Shiqiang
-===== Procedure: =====
+==== Procedure: ====
 # Make enzyme cutting system solution.
@@ Line 2,332: / Line 2,332: @@
 # Go gel electrophoresis.
-===== Results: =====
+==== Results: ====
 [[File:media/image3.png|389x357px]]
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 {{:Team:SUSTech_Shenzhen/wiki-footer}}
 {{:Team:SUSTech_Shenzhen/themeJs}}

Difference between revisions of "Team:SUSTech Shenzhen/Notebook/Biobricks"

Revision as of 01:13, 13 October 2016

Biobricks

Contents

8 Chromoprotein

Plasmid Construction

7.22

Transformation of ①, ③, ⑤

Procedure: IGEM Protocols

7.23

Picking single colonies of ①, ③, ⑤

Procedure:

7.24

Plasmid extraction of ①, ③, ⑤ A and B

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

7.25

Transformation of ②,④,⑥,⑦,⑧,⑨

Procedure: IGEM Protocols

Picking single colonies of ②,④,⑥,⑦,⑧,⑨

Procedure:

7.26

Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group α

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group β

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

7.27

Competent Cell Test

Procedure: IGEM Protocols

Enzyme Digestion and Go Gel Electrophoresis Test of ②,④,⑥,⑦,⑧,⑨

Procedure:

Results:

7.28

Competent Cell Test Colonies Counting

Result:

7.29

Transformation of ⑧,⑩，11

Procedure: IGEM Transformation Protocol

Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑧,⑨

Procedure:

7.30

Picking single colonies of ⑧, ⑩, 11

Procedure:

Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑧,⑨

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results：

Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑨ (Redo)

Procedure:

Plasmid extraction of ⑧,⑩,11

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Enzyme Digestion for Gel Extraction of ⑧,⑩,11

Procedure:

Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑨ (Redo)

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

7.31

Ligation of ⑥+⑤, ⑦+⑤, ⑨+⑤

Procedure:

Go Gel Electrophoresis and Gel Extraction of ⑧, ⑩, 11

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

Transformation of ⑧

Procedure: IGEM Transformation Protocol

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑨+⑤

Procedure:

8.1

Picking single colonies of ⑧

Procedure:

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B

Procedure:

Results:

Put ⑧ A and B at 4℃

8.2

Plasmid extraction of ⑧ A and B