Team:USTC/Notebook/8 5

Modeling

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Date: 8.5

Plasmid Extraction for pGal1-GFP Recorder: Yin Wu and Kaiyue Ma

sample:bacteria containing pGal1-GFP

number	1-1	1-2	1-3	2-1	2-2	2-3
A260/A280	1.88	1.89	1.89	1.81	1.90	1.89
concentration(ng/μL)	196.0	191.9	116.5	250.4	189.5	157.5

Recorder: Kaiyue Ma & Yonghao Liang Transformation of plasmid pSB1A3, YeUGAP

Recorder: Yin Wu and Chengle Zhang PCR of AD and BD

Procedure:

1.Prepare 8 PCR tubes and sequentially add： 22 μL Sterilized ddH₂O, 25 μL 2×HiFi-PCR Master, 1 μL template, 1 μL primer 1, 1 μL primer 2.

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	10 min
step 2	95	1 min
step 3	57	1 min
step 4	72	54 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

(For the two pictures,the left four are AD,the right four are BD.)

4.Then we did gel extration.The result is as follows:

sample	AD4	AD5	BD2	BD4
A260/A280	1.88	1.89	1.90	1.89
concentration(ng/μL)	196.1	84.6	111.1	152.1

Double fast digest of combinant plasmid of pGal1 and GFP Recorder: Ya Jiang and Chengle Zhang

We use the reaction system as follows: 35 μL plasmid, 7 μL Sterilized ddH₂O, 5 μL Greenbuffer, 1.5 μL EcoRI, 1.5 μL PstI.

Agarose gel electrophoresis Result:

图片名称

We did gel extration.The result are as follows:

sample	pGal1-GFP1	pGal1-GFP2
A260/A280	1.97	1.98
concentration(ng/μL)	7.6	8.8

Double fastdigest of AD Recorder: Yu Xie

We use the reaction system as follows: 15 μL plasmid, 1 μL Sterilized ddH₂O, 2 μL fastdigest buffer, 1 μL EcoRI, 1 μL PstI.

Special notes: Use clean water,clean TAE buffer,clean erlenmeyer flask to make superclean gel.

Agarose gel electrophoresis Result:

图片名称

We did gel extration. Special notes: Use wash solution three or four times. If the result is great,we can repeat the elution to get more product.

The result are as follows:

first elution:

sample	AD-1
A260/A280	1.90
A260/A230	0.29
concentration(ng/μL)	118.6

second elution

sample	AD-2
A260/A280	1.82
A260/A230	0.82
concentration(ng/μL)	70.5

third elution

sample	AD-3
A260/A280	1.90
A260/A230	0.44
concentration(ng/μL)	22.0

fourth elution

sample	AD-4
A260/A280	1.76
A260/A230	0.46
concentration(ng/μL)	19.9

Recorder: Yonghao Liang Gel Extraction of PCR product of SUP35

sample	1	2	3	4
A260/A230	0.02	0.09	0.01	0.19
A260/A280	1.93	1.93	1.95	1.92
concentration(ng/μL)	95.8	125.0	14.5	17.4

(sample 3 4 come from the second time we elute the absorbing column 1 and 2)

Date: 8.6

Recorder：Yinchenguang Lyu and Chengle Zhang Double Digestion of BD and combinant plasmid of pGal1&GFP

Reaction system: 16 μL plasmid, 2 μL fastdigest greenbuffer, 1 μL EcoRI, 1 μL PstI.

Agarose gel electrophoresis Result: 图片名称

We did gel extration.The result are as follows:

first elution:

sample	BD	pGal1&GFP
A260/A280	1.90	1.94
A260/A230	0.62	0.94
concentration(ng/μL)	92.9	29.1

second elution:

sample	BD	pGal1&GFP
A260/A280	1.99	1.94
A260/A230	0.27	1.04
concentration(ng/μL)	24.5	12.0

Recorder: Kaiyue Ma PCR of SUP35

Procedure:

1.Prepare 4 PCR tubes and sequentially add： 32.5 μL Sterilized ddH₂O, 10 μL 5X PS Buffer, 4 μL dNTP, 0.5 μL PrimeStar, 1 μL template, 1 μL primer 1, 1 μL primer 2.

2.About primers

primer	1	2
sequence	5'-CCGGAAT TCGCGGCC GCTTCTAGA TGGGCGATT CAAACCAA GGCAACA-3'	5'-TGCACTG CAGCGGCC GCTACTAGT AATCGTTAA CAACTTCG TCATCCACTTCT-3'

3.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	7 min
step 2	95	10 s
step 3	57	15 s
step 4	72	50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

4.Agarose gel electrophoresis Result: 图片名称

5.Conclusion: In the result of agarose gel electophoresis, we can see that the band is at a lower place than where it should be. But we still assume this is the band of SUP35, and keep doing this same experiment to check whether this is the SUP35 or not.

Recorder: Yonghao Liang PCR of SUP35

Procedure:

1.Prepare 4 PCR tubes and sequentially add： 32.5 μL Sterilized ddH₂O, 10 μL 5X PS Buffer, 4 μL dNTP, 0.5 μL PrimeStar, 1 μL template, 1 μL primer 1, 1 μL primer 2.

2.About primers

primer	1	2
sequence	5'-CCGGAAT TCGCGGCC GCTTCTAGA TGGGCGA TTCAAACCA AGGCAACA-3'	5'-TGCACTG CAGCGGCC GCTACTAGT AATCGTT AACAACTTC GTCATCCACT TCT-3'

3.PCR reaction Parameters setting： |stage|temperature|time| |-| |step 1|95|7 min| |step 2|95|10 s| |step 3|57|15 s| |step 4|72|50 s| |step 5|72|10 min| |step 6|4|--| 35 cycles(step 2 ~ step 4)

4.Agarose gel electrophoresis Result:

Plasmid Extraction for sfGFP1-10 **Recorder: Yin Wu,Wenkai Han **

sample:bacteria containing sfGFP1-10

number	1	2	3	4	5	6
A260/A280	1.87	1.88	1.89	1.87	1.87	1.86
concentration(ng/μL)	344.5	282.7	244.5	327.3	380.5	473.6

Agarose gel electrophoresis gel Agarose gel electrophoresis Result: 图片名称

Recorder: Ya Jiang, Yin Wu Transformation of recombinant plasmid of sfGFP1-10 and PYescGAP

Recorder: Yonghao Liang Gel Extraction of PCR product of SUP35

sample	1	2
A260/A280	1.87	1.90
concentration(ng/μL)	90.4	100.5

Recorder: Yonghao Liang & Chenyang Li PCR of SUP35

Procedure:

1.Prepare 4 PCR tubes and sequentially add： 32.5 μL Sterilized ddH₂O, 10 μL 5X PS Buffer, 4 μL dNTP, 0.5 μL PrimeStar, 1 μL template, 1 μL primer 1, 1 μL primer 2.

2.About primers

primer	1	2
sequence	5'-CCGGAAT TCGCGGCC GCTTCTAGA TGGGCGA TTCAAACCA AGGCAACA-3'	5'-TGCACTG CAGCGGCC GCTACTAGT AATCGTT AACAACTTC GTCATCCACT TCT-3'

3.PCR reaction Parameters setting： |stage|temperature|time| |-| |step 1|95|7 min| |step 2|95|10 s| |step 3|57|15 s| |step 4|72|50 s| |step 5|72|10 min| |step 6|4|--| 35 cycles(step 2 ~ step 4)

4.Agarose gel electrophoresis Result: 图片名称

5.Gel Extraction of PCR product of SUP35: |sample|1|2|3|4| |-| |A260/A230|1.59|1.39|0.27|0.43| |A260/A280|1.86|1.86|1.90|1.84| |concentration(ng/μL)|363.2|276.6|120.1|86.2| (sample 3,4 come from the second time we elute the absorbing column 1 and 2)

Recorder：Chengle Zhang Double Digestion of PYescGAP

Reaction system: 16 μL plasmid, 2 μL fastdigest greenbuffer, 1 μL EcoRI, 1 μL PstI.

Agarose gel electrophoresis Result: 图片名称

It shows the DNA travels very slow in the gel because of its high dencity.It tells us not to use the gel which has been put for a really long time.The experiment failed,so we didn't do gel extraction.

Recorder：Chengle Zhang Ligation of PYescGAP and pGal1-GFP,PYescGAP and AD,PYescGAP and BD

Reaction system: 1.5 μL PYescGAP, 2 μL 10× T4 DNA ligase buffer, 0.2 μL T4 DNA ligase, 3.2 μL pGal1-GFP(29.1 ng/μL)/1 μL AD(30.0 ng/μL)/1 μL BD(31.0 ng/μL), 13.1 μL/15.3 μL/15.3 μL nuclease-free water

Mix gently and incubate at 22 degree Celsius for 1 hour.

Then we do transformation of combinant plasmid of PYescGAP and pGal1-GFP, PYescGAP and AD, PYescGAP and BD.

Recorder: Yu Xie Transformation of plasmid containing sfGFP11

Date: 8.7

Plasmid Extraction for pUC57 containing sfGFP11 Recorder: Yu Xie

sample	sfGFP11-5	sfGFP11-6	sfGFP11-7	sfGFP11-8
A260/A280	1.84	1.93	1.71	1.55
A260/A230	1.79	2.51	1.04	0.56
concentration(ng/μL)	49.2	68.7	14.5	36.3

Recorder：Ya Jiang Double Digestion of pSB1A3, pSB1C3-PRsfGFP1-10 and pUC57-sfGFP11

Procedure: In either of the samples of pSB1A3, add nuclease-free water 1 μL 10*Green Buffer 4 μL plasmid 33 μL EcoRI 1 μL PstI 1 μL Mix gently and incubate at 37 degree Celsius for 15 min.

In either of the samples of pSB1C3-PRsfGFP1-10, add nuclease-free water 27 μL 10*Green Buffer 4 μL plasmid 7 μL EcoRI 1 μL PstI 1 μL

In the sample of pUC57-sfGFP11, add nuclease-free water 27 μL 10*Green Buffer 4 μL plasmid 5 μL EcoRI 1 μL XbaI 1 μL Mix gently and incubate at 37 degree Celsius for 15 min. Agarose gel electrophoresis Results: 图片名称

sample	sfGFP11	pSB1A3
A260/A230	0.07	0.08
A260/A280	2.03	1.83
concentration(ng/μL)	14.0	12.5

Plasmid Extraction for pYeUGAP Recorder: Ya Jiang

sample	3-2	4-1	4-2	5-1	5-2	3-1
A260/A280	1.71	1.77	1.87	1.85	1.89	1.86
A260/A230	0.89	1.26	2.37	2.23	2.27	2.14
concentration(ng/μL)	49.2	251.1	84.2	649.3	111.4	260.9

Plasmid Extraction for pSB1A3 Recorder: Ya Jiang

sample	1	2
A260/A280	1.83	1.90
A260/A230	1.94	2.03
concentration(ng/μL)	68.2	49.3

Recorder: Yinchenguang Lyu and Chengle Zhang Double digestion of sup35 Reaction system: 8 μL plasmid, 8 μL nuclease-free water, 2 μL fastdigest greenbuffer, 1.6 μL EcoRI, 0.4 μL SpeI.

Mix gently and incubate at 37 degree Celsius for 30 mins .

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(Transplus 2K),sample1,,sample2,sample3)

We did gel extration.The result are as follows:

sample	1	2	3
A260/A280	1.86	1.82	1.72
A260/A230	0.81	0.42	0.06
concentration(ng/μL)	98.4	73.7	16.5

Recorder：Chengle Zhang Double Digestion of combinant plasmid of PGal1 and GFP

Reaction system: 16.4 μL plasmid, 2 μL fastdigest greenbuffer, 1.2 μL EcoRI, 0.4 μL PstI.

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(Transplus 2K),sample1,sample1,control1,sample2,sample2,control2)

We did gel extration.The result are as follows:

group	1
A260/A280	1.89
A260/A230	0.41
concentration(ng/μL)	30.2

Recorder：Chengle Zhang Colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD

We did colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.Then we did PCR with primer designed by ourselves(biobrick-p/r).The agarose gel electrophoresis result is as follows: 图片名称 (from left to right:AD-1,AD-2,AD-3,AD-control,BD-1,BD-2,BD-3,BD-control) It shows that the colonies we picked are not successful ones.

Recorder: Yu Xie & Yinchenguang Lyu

Double digestion of plasmid containing sfGFP11 and pSB1C3-PRsfGFP1-10 Reaction system:

Sample	24μL sfGFP11-1	24μL sfGFP11-2	20μL sfGFP11-3	18μL pSB1C3-PRsfGFP1-10-1	18μL pSB1C3-PRsfGFP1-10-2	8μL pSB1C3-PRsfGFP1-10-3
nuclease-free water(μL)	0	0	4	6	6	8
fastdigest buffer(μL)	3	3	3	3	3	2
PstI(μL)	1.5	1.5	1.5	1.5	1.5	1
XbaI(μL)	1.5	1.5	1.5	0	0	0
EcoRI(μL)	0	0	0	1.5	1.5	1
total(μL)	30	30	30	30	30	20

Mix gently and incubate at 37 degree Celsius for 30 mins .

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(Transplus 2K),sample1,,sample2,sample3,control-1,sample4,sample5,sample6,control-2)

We did gel extration.The result are as follows:

sample	pSB1C3-PRsfGFP1-10-1	pSB1C3-PRsfGFP1-10-2
A260/A230	0.56	0.33
A260/A280	1.87	1.79
concentration(ng/μL)	202.7	60.7

(sample 2 come from the second time we elute the absorbing column 1)

Recorder: Kaiyue Ma Plasmid Extraction for pSB1C3 and YeTGAP

Result:

sample	pSB1C3-1	pSB1C3-2	YeTGAP	YeTGAP	YeTGAP
A260/A280	1.91	1.85	1.89	1.86	1.88
concentration(ng/μL)	300.0	580.4	993.5	593.0	968.1

Recorder: Chengle Zhang Transformation of plasmid pSB1C3,combinant plasmid of PYescGAP and pGal1-GFP, PYescGAP and AD,PYescGAP and sfGFP1-10

We did transformation of plasmid pSB1C3,combinant plasmid of PYescGAP and pGal1-GFP, PYescGAP and AD,PYescGAP and sfGFP1-10. After 12 hours,we check the plate.Only on PYescGAP and pGal1-GFP plate grows bacteria.

Recorder: Yu Xie Transformation of plasmid pSB1A3

Date: 8.8 Recorder: Wenkai Han PCR of sfGFP11

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1-4
Sterilized ddH₂O	32.5 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL
dNTP Mixture (2.5 mM each)	4 μL
Template (sfGFP)	1 μL
standard-biobrick-p	1 μL
standard-biobrick-r	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL
total	50 μL

2.PCR reaction

Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	10 s
step 3	55	15 s
step 4	72	12 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

The agarose gel electrophoresis result is as follows: 图片名称

Recorder: Yu Xie Ligation of PYescGAP and PRsfGFP1-10

Material: 1. double digestion product of PYescGAP 2. double digestion product of PRsfGFP1-10 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: 1.Add to either of samples:

Sample	volume
PYescGAP (cut)	1.5μL
PRsfGFP1-10	0.5μL
nuclease free water	15.7μL
10* T4 DNA Ligase Buffer	2.0μL
T4 DNA Ligase	0.3μL
total	20μL

2.Mix gently and incubate at 22 degree Celsius for 1 hour.

Recorder: Yonghao Liang & Yu Xie Double digestion pSB1A3

Reaction system:

Agarose gel electrophoresis Result:

Then we did gel extraction,the parameters of product are as follows:

Plasmid Extraction for pSB1A3 Recorder: Yonghao Liang

sample	1	2	3	4
A260/A280	1.91	1.94	1.91	1.89
A260/A230	1.99	2.06	1.22	1.72
concentration(ng/μL)	84.6	74.7	86.9	104.4

Double fastdigest of PSB1A3 Recorder: Yu Xie & Yonghao Liang

We use the reaction system as follows: 16 μL plasmid, 2 μL fastdigest buffer, 1 μL EcoRI, 1 μL PstI, 0 μL Sterilized ddH₂O.

Agarose gel electrophoresis Result: 图片名称 (PS: 1 to 4 are the digest products, 5 is the control group) We did gel extration.

The result are as follows:

sample	1
A260/A280	2.12
A260/A230	0.04
concentration(ng/μL)	34.6

Recorder: Kaiyue Ma Transformation of pYeWGAP-pGAL-GFP, pYeWGAP-AD and pYeWGAP-BD Bacteria: top 10 Plates: Ap+ 50 μg/mL

Recorder：Chenyang Li,Yinchenguang Lyu and Chengle Zhang Check again for the last picking colony of combinant plasmid of PYescGAP and AD,PYescGAP and BD

The agarose gel electrophoresis result is as follows: 图片名称 (from left to right:AD-cut,AD-PCR,AD-1-1,AD-1-2,AD-1-3,AD-2-1,AD-2-2,AD-2-3,BD-cut,BD-PCR,BD-1-1,BD-1-2,BD-1-3,BD-2-1,BD-2-2,BD-2-3) It still shows that the colonies we picked are not successful ones.

Recorder：Chengle Zhang Colony picking of combinant plasmid of PYescGAP and pGal1&GFP,PYescGAP and AD,PYescGAP and BD

We did colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD again.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.Then we did PCR with primer designed by ourselves(biobrick-p/r). The agarose gel electrophoresis result is as follows:

Recorder：Chengle Zhang Ligation of PYescGAP and pGal1-GFP,PYescGAP and AD,PYescGAP and BD again

Reaction system: 1.5 μL PYescGAP, 2 μL 10× T4 DNA ligase buffer, 0.2 μL T4 DNA ligase, 3.2 μL pGal1-GFP(29.1 ng/μL)/1 μL AD(30.0 ng/μL)/1 μL BD(31.0 ng/μL), 13.1 μL/15.3 μL/15.3 μL nuclease-free water

Mix gently and incubate at 22 degree Celsius for 1 hour.

Then we do transformation of combinant plasmid of PYescGAP and pGal1-GFP, PYescGAP and AD, PYescGAP and BD.

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