8.16 Recorder: Dong Yan & Kaiyue Ma

Sample:bacteria containing recombinant plasmid pYeT containing sg11

Number	1	2	3	4	5	6	7	8
A260/A280	1.89	1.86	1.76	1.88	1.87	1.81	1.86	1.88
A260/230	2.27	2.02	1.23	2.12	2.23	1.45	1.82	2.25
concentration(ng/μL)	159.3	113.5	265.7	46.8	130.5	189.3	59.7	169.3

Double digestion of AD, BD and YeUGAP Recorder: Yinchenguang Lyu

Reaction system:

AD & BD: 4 μL template 1.35 μL EcoRI 2 μL PstI 2 μL 10× FastDigest Buffer 10.65 Sterilized ddH₂O

YeUGAP: 5 μL template 1 μL EcoRI 0.35 μL PstI 2 μL 10× FastDigest Buffer 11.65 Sterilized ddH₂O

Gel Extraction for the product of the double digestion Recorder: Yinchenguang Lyu

sample	AD	BD	YeUGAP
A260/A280	1.89	1.84	1.86
A260/230	0.48	0.85	0.79
concentration(ng/μL)	13.1	25.1	33.9

Recorder: Chenyang Li Plasmid Extraction for YeWGAP

sample	08171001	08171002	08171003	08171004	08171005	08171006
A260/280	1.89	1.83	1.80	1.70	2.88	1.89
A260/230	2.36	1.83	1.47	0.99	2.07	2.14
concentration(ng/μL)	916.3	359.3	300.5	193.6	53.6	132.2

Ligation of YeUGAP and AD Recorder: Yinchenguang Lyu

3 μL YeUGAP 3 μL AD 0.4 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 11.6 μL ddH₂O 20 μL in total.

Double digestion of AD,BD and PYU Recorder:Chengle Zhang

Sample	AD(0230)	BD(0231)	PYU(0232)
A260/A280	1.97	1.83	1.89
A260/A280	0.12	0.41	0.75
concentration(ng/μL)	24.8	62.5	105.4

Plasmid Extraction for sfGFP1-10 Recorder: Xuefeng Meng

Sample:bacteria containing recombinant plasmid pYeT containing sfGFP1-10

Number	1	2	3	4
A260/A280	1.93	1.89	1.92	1.93
A260/230	2.04	1.46	1.38	2.04
concentration(ng/μL)	43.2	19.7	49.3	20.8

**Recorder：Chenyang Li ** Double Digestion of YeWGAP, ADU, BDW

Procedure:

number	YeWGAP18171001	YeWGAP18171002	YeWGAP18171003	YeWGAP18171004	ADU1001	ADU1002	BDW1003	BDW1004
sample	YeWGAP08171001	YeWGAP08171002	YeWGAP08171003	YeWGAP08171004	ADU1	ADU2	BDW3	BDW4
nuclease-free water(μL)	8.4	76	7.3	5.4	0	4	0	0
10*Green Buffer(μL)	1	1	1	1	1	1	1	1
plasmid(μL)	0.6	1.4	1.7	2.6	8	4	8	8
EcoRI(μL)	0.75	0.75	0.75	0.75	0.75	0.75	0.75	0.75
PstI(μL)	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25

Mix gently and incubate at 37 degree Celsius for 15 min.

result: (from left to right: Trans 2K plus 2(contain Gelred),08171001,18171001,18171002,08171003,18171003,18171004,ADU1,ADU1001,ADU2,ADU1002,BDW3,BDW1003,BDW4，BDW1004.)

Ligation of pSB1C3 and AD Recorder: Yinchenguang Lyu

4 μL pSB1C3 2.8 μL AD 0.4 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 10.8 μL ddH₂O 20 μL in total.

DATE:8.18

**Recorder：Chenyang Li ** Double Digestion of YeWGAP,BD

Procedure:

number	YeWGAPD8181001	YeWGAPD8181006	BDD8181001	BDD8181002
sample	YeWGAP08171001	YeWGAP08171006	BD(336)	BD(336)
nuclease-free water(μL)	15.6	9	13.6	13.6
10* Fast Digest Buffer(μL)	2	2	2	2
plasmid(μL)	1	7.6	3	3
EcoRI(μL)	1	1	1	1
PstI(μL)	0.4	0.4	0.4	0.4

20 μL in total. Mix gently and incubate at 37 degree Celsius for 15 min.

result: (from left to right: Trans 2K plus 2(contain Gelred),YewGAP08171001,D8181001,YewGAP08171006,D8181006,BD(363),BDD8181001,BDD8181002.)

Plasmid Extraction for YeLGAP LYH Recorder: Dong Yan

Number 1 2 3 4 A260/A280 1.84 1.87 1.91 1.87 A260/230 2.30 2.34 2.40 2.28 concentration(ng/μL) 634.4 459.6 806.8 387.5

Recorder: Chenyang Li Gel Extraction of double digestion product of YeWGAP and BD

sample	YeWGAPD8181001	BDD8181001
A260/A280	1.68	1.84
A260/A230	0.2	0.15
concentration(ng/μL)	19.8	21.4
volume(μL)	50	25

PCR of AD, BD and sup35 Recorder: Yinchenguang Lyu

sample	AD	BD	sup35
nuclease-free water(μL)	22	22	32.5
2×HiFi-PCR Master(μL)	25	25	0
template(μL)	1	1	1
5×PrimeStar Buffer(μL)	0	0	10
dNTP(μL)	0	0	4
PrimeStar	0	0	0.5
Biobrick Primer-r	2	2	1
Biobrick Primer-p	2	2	1

Purification for the product of the PCR Recorder: Yinchenguang Lyu

results

Number	AD1	AD2	BD1	BD2	sup35-1	sup35-2	sup35-3	sup35-4
A260/A280	1.83	1.83	1.85	1.86	1.80	1.72	1.84	1.78
A260/A230	1.83	2.20	2.10	2.18	1.05	0.85	1.08	1.27
concentration(ng/μL)	212.3	200.6	186.0	230.9	12.3	21.2	21.5	20.9

Ligation of YeWGAP and BD **Recorder: Chenyang Li **

Procedure: 5 μL YeWGAP 2 μL BD 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 10.8 μL ddH₂O 20 μL in total. Mix gently and incubate at 22 degree Celsius for an hour. The number of the product: YBL8181001

Plasmid Extraction for YeWGAP Recorder: Junjie Zeng and Chenyang Li

Sample:bacteria containing recombinant plasmid YeWGAP

Number	YeWGAP08181007	YeWGAP08181008	YeWGAP08181009	YeWGAP08181010
A260/A280	1.81	1.86	1.86	1.85
concentration(ng/μL)	740.5	841.2	725.0	695.9

DATE:8.19

**Recorder：Chenyang Li ** Double Digestion of YeWGAP,BD

Procedure:

number	1	2	3	4	5	6	7
sample	YeWGAP08171001	YeWGAP08171001	YeWGAP08171001	YeWGAP08171001	BD(336)	BD(336)	BD(230.9)
nuclease-free water(μL)	11	11	11	11	12	14.5	6.4
10* Fast Digest Buffer(μL)	2	2	2	2	2	2	2
plasmid(μL)	5	5	5	5	5	3	10
EcoRI(μL)	1.5	1.5	1.5	1.5	0.7	0.3	1.2
PstI(μL)	0.5	0.5	0.5	0.5	0.3	0.2	0.4

20 μL in total. Mix 1,2,3,4,5,6 gently and incubate at 37 degree Celsius for 45 min. Mix 7 gently and incubate at 37 degree Celsius for 30 min.

result: (from left to right: Trans 2K plus 2(contain Gelred),YewGAP08171004,1,2,3,4,5,6,7.)

Plasmid Extraction for YeWGAP Recorder: Junjie Zeng and Chenyang Li

Sample:bacteria containing recombinant plasmid YeWGAP

Number	YeWGAP08181011	YeWGAP08181012	YeWGAP08181013	YeWGAP08181014
A260/A280	1.89	1.87	1.88	1.89
concentration(ng/μL)	818.6	416.9	807.4	900.6

Plasmid Extraction for YeLGAP Recorder: Xuefeng Meng

Sample:bacteria containing plasmid YeLGAP

Number	1	2	3	4	5	6	7	8
concentration(ng/μL)	128.1	228.7	60.5	67.0	398.3	291.6	80.8	101.1
A260/A280	1.83	1.73	1.93	1.93	1.86	1.82	1.90	1.86
A260/230	2.00	1.12	2.75	2.69	2.39	1.84	2.48	1.99

Recorder:Weijie Jin and Chenyang Li Gel Extraction of double digestion product of YeWGAP and BD

sample	YeWGAPD8191002	BDD8191002
A260/A280	1.86	1.93
A260/A230	0.72	0.14
concentration(ng/μL)	315.1	52.5

Plasmid Extraction for SBAD-2 4 5 Recorder: Dong Yan

Number	-2-1	-2-2	-2-3	-4-1	-4-2	-4-3	-5-1	-5-2	-5-3
A260/A280	1.89	1.90	1.86	1.83	1.81	1.92	1.89	1.93	1.82
A260/230	2.29	2.19	1.56	1.56	1.88	2.18	1.71	2.20	1.33
concentration(ng/μL)	77.3	72.4	61.7	120.5	77.4	57.6	81.7	79.7	68.4

The absorption peaks are normal, so the poor concentration may due to lack of bacteria growing time.

Double digestion of PSB1C3 Recorder: Yinchenguang Lyu

6μL PSB1C3 1μL EcoRI 0.35μL PstI 2μL 10×Fast Digest Buffer 13.65μL ddH₂O 20μL in total.

result:

Gel Extraction of double digestion product of PSB1C3 Recorder: Yinchenguang Lyu

sample	PSB1C3:0802
A260/A280	1.86
A260/A230	1.28
concentration(ng/μL)	119.3

Ligase of PSB1C3 and BD Recorder: Yinchenguang Lyu

1μL PSB1C3 4μL BD 2μL 10×T4 DNA Ligase Buffer 0.4μL T4 DNA Ligase 12.6μL ddH₂O

DATE:8.20

Ligation of YeWGAP and BD **Recorder: Chenyang Li **

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Mix gently and incubate at 22 degree Celsius for an hour.

The number of the product: YBL8201002 and YBL8201003

Then we do transformation.

**Recorder: Chenyang Li ** PCR of Sup35

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	sup35
nuclease-free water(μL)	30.5
5×PrimeStar Buffer(μL)	10
dNTP(μL)	4
template(μL)	4
PrimeStar	0.5
Primer1	0.5
Primer2	0.5

50 μL in total.

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95	7 min
Duration	95	10s
Anneal	57	15s
Extend	72	50s
Post-Extend	72	10 min
Final	4	--

35 cycles(Duration ~ Extend)

Purification for the product of the PCR of Sup35 Recorder: Chenyang Li

results

Number	Sup35P8201001	Sup35P8201002	Sup35P8201003	Sup35P8201004
A260/A280	1.87	1.88	1.86	1.87
A260/A230	2.29	2.29	1.91	2.13
concentration(ng/μL)	170.2	302.3	334.6	301.0

**Recorder：Chenyang Li ** Double Digestion of YeWGAP,BD

Procedure:

number	1	2	3	4
sample	Sup35P8201002	Sup35P8201002	Sup35P8201004	Sup35P8201004
nuclease-free water(μL)	4	7.8	4	7.8
10* Fast Digest Buffer(μL)	2	2	2	2
plasmid(μL)	10	7	10	7
EcoRI(μL)	3	2.4	3	2.4
Bcul(μL)	2.4	0.8	2.4	0.8

20 μL in total. Mix 1,2,3,4gently and incubate at 37 degree Celsius for 15 min.

result: (from left to right: Trans 2K plus 2(contain Gelred),1,2,3,4,PSB1C3+AD1,PSB1C3+AD2,PSB1C3+AD3,PSB1C3+AD4,)

DATE:8.22

Ligation of YeWGAP and BD **Recorder: Chenyang Li **

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Mix gently and incubate at 22 degree Celsius for an hour.

The number of the product: YBL8201004. Then we do transformation.

PCR of sup35 **Recorder: Yinchenguang Lyu **

21μL ddH₂O 25μL 2×HiFi PCR Master 2μL template 1μL primer1 1μL primer2

stage	temperature	time
step 1	95	5 min
step 2	95	50 s
step 3	56	1 min
step 4	72	1 min 50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

DATE 8.23

Plasmid Extraction for ligased PSB1C3 and AD Recorder: Yinchenguang Lyu

results

sample	1	2	3	4
A260/A280	1.81	1.77	1.70	1.57
A260/A230	1.64	1.43	0.79	0.78
concentration(ng/μL)	175.9	204.4	216.3	346.1

Recorder：Yu Xie Colony picking of plasmid pUC57 containing sfGFP11

We did colony picking of plasmid pUC57 containing sfGFP11. After colony picking, we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Recorder: Junjie Zeng & Yu Xie PCR of sup35

Procedure:

1.Prepare 8 PCR tubes and sequentially add：

sample	1,2,3,4,5,6,7,8
Sterilized ddH2O	9 μL
2×HiFi-PCR Master	12.5 μL
Template	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95℃	10 min
Duration	95℃	1 min
Anneal	58℃	1 min
Extend	72℃	1 min 50 s
Post-Extend	72℃	10 min
Final	4℃	--

30 cycles(Duration ~ Extend)

Recorder: Yu Xie Double digestion of sfGFP11

Recorder: Yu Xie Double digestion of sup35

DATE：8.24

Recorder: Yu Xie Plasmid Extraction for sfGFP11

Recorder: Yu Xie PCR of sup35

Procedure:

1.Prepare 8 PCR tubes and sequentially add：

sample	1,2,3,4,5,6,7,8
Sterilized ddH2O	9 μL
2×HiFi-PCR Master	12.5 μL
Template	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95℃	10 min
Duration	95℃	1 min
Anneal	58℃	1 min
Extend	72℃	1 min 50 s
Post-Extend	72℃	10 min
Final	4℃	--

30 cycles(Duration ~ Extend)

Recorder: Yu Xie Double digestion of sup35

Recorder：Yu Xie Ligation of sup35 and pUC57-sfGFP11

Recorder: Yu Xie Transformation of combinant plasmid pUC57 containing sup35-sfGFP11

Recorder: Yu Xie PCR of sup35

Procedure:

1.Prepare 8 PCR tubes and sequentially add：

sample	1,2,3,4,5,6,7,8
Sterilized ddH2O	9 μL
2×HiFi-PCR Master	12.5 μL
Template	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95℃	10 min
Duration	95℃	1 min
Anneal	58℃	1 min
Extend	72℃	1 min 50s
Post-Extend	72℃	10 min
Final	4℃	--

30 cycles(Duration ~ Extend)

Recorder：Yu Xie Colony picking of sfGFP11

Recorder: Chenyang Li PCR of pYeWGAP-BD

Procedure:

3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add： 4 μL Sterilized ddH₂O, 6.5 μL 2XHiFi Pfu, 0.5 μL primer F, 0.5 μL primer R, 1 μL DNA template.

4.PCR reaction Parameters setting：

stage	temperature	time
step 1	95℃	10 min
step 2	95℃	30 s
step 3	58℃	45 s
step 4	72℃	45 s
step 5	72℃	10 min
step 6	4℃	--

30 cycles(step 2 ~ step 4)

results:

(from left to right: Trans 2K plus 2(contain Gelred), 1, 2, 3, 4)

Plasmid Extraction for ligased YeWGAP and BD Recorder: Chenyang Li

results

sample	YBE8241001	YBE8241002	YBE8241003	YBE8241004
A260/A280	1.88	1.86	1.89	1.88
A260/A230	2.27	1.92	2.22	2.22
concentration(ng/μL)	203.1	174.7	181.3	177.8

DATE：8.24 Recorder: Chenyang Li PCR of pYeWGAP-BD

Procedure:

3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add： 3 μL Sterilized ddH₂O, 5 μL 2XHiFi Pfu, 0.5 μL primer F, 0.5 μL primer R, 1 μL DNA template.

4.PCR reaction Parameters setting：

stage	temperature	time
step 1	95℃	10 min
step 2	95℃	30 s
step 3	58℃	45 s
step 4	72℃	45 s
step 5	72℃	10 min
step 6	4℃	--

30 cycles(step 2 ~ step 4)

results:

(from left to right: Trans 2K plus 2(contain Gelred),YBE8241001，1，YBE8241002，2，YBE8241003，3，YBE8241004，4，)

DATE:8.25

Recorder: Yu Xie Plasmid Extraction for sfGFP11

Recorder: Yu Xie Transformation of combinant plasmid pUC57 containing sup35-sfGFP11

Recorder: Yu Xie PCR of sup35

Procedure:

1.Prepare 8 PCR tubes and sequentially add：

sample	1,2,3,4,5,6,7,8
Sterilized ddH2O	9 μL
2×HiFi-PCR Master	12.5 μL
Template	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95℃	10 min
Duration	95℃	1 min
Anneal	58℃	1 min
Extend	72℃	1 min 50 s
Post-Extend	72℃	10 min
Final	4℃	--

30 cycles(Duration ~ Extend)

Recorder：Yu Xie Colony picking of sup35-sfGFP11

Ligation of YeWGAP and BD **Recorder: Chenyang Li **

Procedure: 9 μL YeWGAPD8201002 3.3 μL BDD8201002 0.2 μL T4 DNA ligase 2 μL 10×T4 DNA Ligase Buffer 5.5 μL ddH₂O 20 μL in total.

Mix gently and incubate at 22 degree Celsius for an hour.

The number of the product: YBL8201005, YBL8201006, YBL8201007, YBL8201008, YBL8201009.

Recorder: Chenyang Li PCR of pYeWGAP-BD

Procedure:

3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add： 4 μL Sterilized ddH₂O, 5 μL 2XHiFi Pfu, 0.5 μL primer F, 0.5 μL primer R, 0.5 μL DNA template.

4.PCR reaction Parameters setting：

stage	temperature	time
step 1	95℃	10 min
step 2	95℃	30 s
step 3	58℃	45 s
step 4	72℃	45 s
step 5	72℃	10 min
step 6	4℃	--

30 cycles(step 2 ~ step 4)

results:

(from left to right: Trans 2K plus 2(contain Gelred),YBE8241001，1，YBE8241002，2，YBE8241003，3，YBE8241004，4，YeWGAP08171001，YeWGAPD8191002，BDD8191002，YBL8201005，Sterilized ddH₂O)

Date: 8.26

Recorder: Yu Xie PCR of sfGFP1-10

Recorder: Yu Xie Double Digestion of sfGFP1-10

Recorder: Yu Xie Double Digestion of pSB1C3

Date: 8.27

Recorder: Yu Xie Ligation of pSB1C3 and sfGFP1-10

Recorder: Yu Xie Transformation of combinant plasmid pSB1C3 containing sfGFP1-10

Recorder：Yu Xie Colony picking of combinant plasmid pSB1C3 containing sfGFP1-10

Recorder: Yu Xie PCR of sfGFP1-10

Recorder: Yu Xie Double Digestion of sfGFP1-10

Plasmid Extraction for plasmid C3BD35 Recorder: Xuefeng Meng

results

sample	1	2	3
A260/A280	2.02	1.90	1.91
A260/A230	1.19	1.07	1.31
concentration(ng/μL)	17.0	15.5	27.9

PCR of sfGFP11 Recorder: Xuefeng Meng results Date: 8.28

Recorder: Yu Xie Plasmid Extraction for pSB1C3 containing sfGFP1-10

Recorder: Yu Xie PCR of bacteria which have recombinant plasmid pSB1C3 containing sfGFP1-10

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1,2,3,4
Sterilized ddH2O	9 μL
2×HiFi-PCR Master	12.5 μL
Template(bacteria)	1.5 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

2.PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95	10 min
Duration	95	1 min
Anneal	58	1 min
Extend	72	1 min 50s
Post-Extend	72	10 min
Final	4	--

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: succeed!

Recorder: Yu Xie PCR of recombinant plasmid pSB1C3 containing sfGFP1-10

Procedure:

1. Prepare 4 PCR tubes and sequentially add：

sample	1,2,3,4
Sterilized ddH2O	9.5 μL
2×HiFi-PCR Master	12.5 μL
Template(plasmid)	1 μL
Primer 1	1 μL
Primer 2	1 μL
total	25 μL

1. PCR reaction Parameters setting：

stage	temperature	time
Pre-Duration	95	10 min
Duration	95	1 min
Anneal	58	1 min
Extend	72	1 min 15s
Post-Extend	72	10 min
Final	4	--

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: succeed!

Date: 8.29

Colony PCR to examine ADW Recorder: Yinchenguang Lyu

6.25 μL 2×Taq PCR Master Mix 2 μL culture containing Ecoli 0.4 μL bio-p 0.4 μL M13-f 3.45 μL ddH₂O

Double Digest to examine ADW and C3AD35 Recorder: Yinchenguang Lyu

result(along with that of the former part)

(from left to right: Trans 2K plus 2(contain Gelred), digested-ADW1, digested-ADW2, C3AD35(1), C3AD35(2), ADW1, ADW2, ADW3, ADW4, ADW5, ADW6)

Date：8.30

Plasmid extraction of pGAL1(site mutation) Recorder: Xuefeng Meng

sample	1	2	3	4
concentratin(ng/μL)	379.6	232.9	277.2	375.4
A260/A280	1.81	1.85	1.90	1.90
A260/A230	1.42	1.72	2.33	2.31