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Date: 7.29

Recorder: Kaiyue Ma

Amplification of bacteria containing sup35

  1. Add 2 μL bacteria to 200 μL SOC liquid medium.
  2. Divide it into two packs.
  3. Coat them separately on two kn+ plates.

Colony picking

  1. Pick 4 colonies of a plate containing sup35gene in 4 test tubes (5 mL LB liquid medium, Kana+).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder： Yonghao Liang Purification of PCR product of SUP35 We have 50 μL solution in each tube to be purified by PCR purification kit (bought from Sangon Biotech). And the procedure is showed below:

  1. Add 250 μL Buffer B3 to the 50 μL solution and mix it up. Add it to an adsorption column.
  2. 11,000 rpm centrifuge for 30 s, and then discard the filtrate.
  3. Add 500 μL Wash Solution, 12,000 rpm centrifuge for 30 s, and then discard the filtrate.
  4. Repeat last process.
  5. Centrifuge the empty column at 12,000 rpm for 1 min.
  6. Lie the column still for 10 min.
  7. Put the column to an 1.5 ml EP tube, add 25 μL ddH2O(55°C), stand them still for 10 min. Centrifuge at 12,000 rpm for 1 min.

Results:

sample 1 2 3 4
A260/A280 1.86 1.85 1.89 1.88
Concentration(ng/μl) 151.7 80.4 111.7 132.7

Recorder：Xuefeng Meng, Chengle Zhang Double Digestion of Sup35 Materials: 1. Sup35 gene () 2. Restriction enzyme (Fastdigest) XbaI, PstI and 10× Green Buffer(bought from Thermo Fisher Scientific) 3. Nuclease-free water 4. Trans2K DNA Marker

Procedure: Add the materials into new EP tubes in the order of following table respectively.

sample 8 μL Sup35-1(278.5 ng/μL) 10 μL Sup35-2(36.2 ng/μL)
nuclease free water(μL) 8 6
10×Green Buffer(μL) 2 2
PstI(μL) 0.5 0.5
XbaI(μL) 0.5 0.5
total(μL) 20 20

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

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of digestion of Sup35 (from left to right:marker,blank,Sup35-1,blank,Sup35-2)

We cut off the Gel part which might contain the target band and store them at 4 degree Celsius.

Recorder：Yin Wu Double Digestion of BBa_K165002 (Kozak sequence (yeast RBS))

Procedure: In either of the samples, add nuclease-free water 14 μL 10*Green Buffer 4 μL plasmid 20 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

Recorder：Chengle Zhang Double Digestion of Sup35 Materials: 1. Sup35 gene () 2. Restriction enzyme (Fastdigest) XbaI, PstI and 10×Green Buffer(bought from Thermo Fisher Scientific) 3. Nuclease-free water 4. Trans2K DNA Marker

Procedure: Add the materials into new EP tubes in the order of following table respectively.

sample 16 μL Sup35-1(151.7 ng/μL) 16 μL Sup35-2(132.7 ng/μL)
10×Green Buffer(μL) 2 2
PstI(μL) 1 1
XbaI(μL) 1 1
total(μL) 20 20

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

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of digestion of Sup35 (from left to right:marker,control,Sup35-1,Sup35-2) (control group contains 2 μL Sup35-1,8 μL nuclease-free water and 2 μL 6× loading buffer)

We cut off the Gel part which might contain the target band and store them at 4 degree Celsius.

Recorder: Yin Wu Gel Extraction of digestion product of yeast RBS

Procedure: 1. Cut off the Gel part containing the target band. Get 4 tubes of digestion product. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of digestion product.

sample 1 2
A260/A280 2.04 failed
concentration(ng/μL) 34.3 failed

The data of sample 2 are strange, showing that there are some problems, eg. the adsorption column is broken, so I discard sample 2.

Recorder: Yu Xie Plasmid Extraction for pGAL1-GFP

Materials: 1. 5 mL LB medium containing recombinant plasmid of pGal1 and GFP

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample pGAL1-GFP-α pGAL1-GFP-β pGAL1-GFP-γ pGAL1-GFP-δ
A260/A280 1.87 1.87 1.87 1.88
concentration(ng/μL) 172.4 129.4 127.7 175.9

Recorder: Ya Jiang Plasmid Extraction for sfGFP1-10 expression plasmids

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample sfGFP1-10-1 sfGFP1-10-2 sfGFP1-10-3 sfGFP1-10-4
A260/A280 1.91 1.91 1.93 2.03
concentration(ng/μL) 64.6 47.5 27.8 16.8

Recorder: Kaiyue Ma Standardization of sup35

Experimental materials 1. PCR product of sup35. 2. Primer: standard-sup35-p, standard-sup35-r. Designed by ourselves, synthesized by Sangon Biotech; 3. Sterilized ddH2O; 4. 5×PrimeSTAR Buffer (Mg2+Plus), dNTP Mixture (2.5 mM each), PrimeSTAR HS DNA Polymerase (2.5 U/μL). All bought from TaKaRa, Code No. R010A.

About primers

primer 1 2
sequence 5'-CCGGAATTCGCG
GCCGCTTCTAGA
TGTCGGATTCAA
ACCAGGGCAACA-3'		 5'-TGCACTGCAGCG
GCCGCTACTAGT
AATCATTCACCA
CCTCATCGTCAA
CTTC-3'

Procedure:

1.Prepare 4 PCR tubes(each sample is put into 2 tubes)and sequentially add：

sample
Sterilized ddH2O 32.5 μL
5×PrimeSTAR Buffer (Mg2+Plus) 10 μL
dNTP Mixture (2.5 mM each) 4 μL
Template(PCR product 189.1ng/μL) 1 μL
standard-sup35-p 1 μL
standard-sup35-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 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 --

30 cycles(step 2 ~ step 4)

Recorder: Yinan Chen Plasmid Extraction for AP+ GFP and sup35-

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O (60℃ preheated), 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of plasmids.

sample AP+ GFP 1 AP+ GFP 2 AP+ GFP 3 AP+ GFP 4 AP+ GFP 5 AP+ GFP 6 AP+ GFP 7 AP+ GFP 8 sup35- 1 sup35- 2 sup35- 3 sup35- 4 sup35- 5 sup35- 6 sup35- 7 sup35- 8
A260/A280 1.97 1.85 1.94 1.90 1.99 1.99 1.98 1.97 2.10 2.06 1.93 2.04 2.10 2.11 2.11 2.02
concentration(ng/μL) 28.3 33.2 32.2 39.1 32.1 27.8 33.4 30.8 18.5 20.8 24.9 21.3 29.9 16.3 17.8 23.0

Recorder: Yin Wu Ligation of pTEF2 and yeast RBS

Material: 1. double digestion product of pTEF2 2. double digestion product of yeast RBS 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: Add to either of samples: 7 μL pTEF2 3 μL yeast RBS 2 μL 10* T4 DNA Ligase Buffer 8 μL nuclease free water 0.25 μL T4 DNA Ligase

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

Recorder: Ya Jiang, Yin Wu Transformation of recombinant plasmid of pTEF2 and yeast RBS

Materials(all made by ourselves): 1. recombinant plasmid of pTEF2 and yeast RBS 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet the recombinant plasmid into competent cell tubes, 10 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 200 µL of SOC media per tube, and incubate at 37°C for 1 hours. 7. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 150 μL supernatant liquid and resuspend the bacteria. 9. Coat plate: add the solution to 2 plates (Cm+) and spread it, respectively, then cultivate it overnight.

Recorder: Kaiyue Ma Agarose Gel Electrophoresis of Standardization sup35 PCR Production 图片名称

Results of gel extraction of standard sup35

1 2 3 4
concentration(ng/μL) 12.9 9.9 13.1 26.8
A260/A280 2.09 2.15 1.89 2.13

I am completely confused. WHY? I DON'T UNDERSTAND! Why can't I extract ENOUGH sample from the gel? How can I manage it. If there is a god of gel extraction, he must be mocking at me.

Amplification of bacteria containing sup35

  1. Add 0.5 μL bacteria to 1000 μL SOC liquid medium.
  2. Coat 20 μL of that solution on a kn+ plate.

Recorder: Xuefeng Meng Standardization of sup35

Experimental materials 1. PCR product of sup35, plasmid containing sup35. 2. Primer: standard-sup35-p, standard-sup35-r. Designed by ourselves, synthesized by Sangon Biotech; 3. Sterilized ddH2O; 4. 5×PrimeSTAR Buffer (Mg2+Plus), dNTP Mixture (2.5 mM each), PrimeSTAR HS DNA Polymerase (2.5 U/μL). All bought from TaKaRa, Code No. R010A. 6. 2×HiFi-PCR Master.

About primers

primer 1 2
sequence 5'-CCGGAATTCGCG
GCCGCTTCTAGA
TGTCGGATTCAA
ACCAGGGCAACA-3'		 5'-TGCACTGCAGCG
GCCGCTACTAGT
AATCATTCACCA
CCTCATCGTCAA
CTTC-3'

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample 1 (plasmid 7.10ng/μL) 2 (plasmid 0.710ng/μL)
Sterilized ddH2O 32.5 μL 32.5 μL
5×PrimeSTAR Buffer (Mg2+Plus) 10 μL 10 μL
dNTP Mixture (2.5 mM each) 4 μL 4 μL
Template 1 μL 1 μL
standard-sup35-p 1 μL 1 μL
standard-sup35-r 1 μL 1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl) 0.5 μL 0.5 μL
total 50 μL 50 μL
sample 3 (PCR product 23.39ng/μL) 4 (PCR product 2.339ng/μL)
Sterilized ddH2O 22 μL 22 μL
2×HiFi-PCR Master 25 μL 25 μL
Templete 1 μL 1 μL
Primer 1 1 μL 1 μL
Primer 2 1 μL 1 μL
total 50 μL 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 50 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

results failed. sample: from left to right: marker, sample 1,2,3,4(50 μL PCR product+10 μL loading buffer)

Recorder: Chengle Zhang Check for digestion enzyme: EcoRI,PstI,SpeI and XbaI

Materials: 1. pGal1 gene in plasmid BBa-J63010(from iGEM kit plate) 2. Restriction enzyme SpeI,PstI,XbaI and 10×Tango Buffer(from Thermo Fisher Scientific) 3. Nuclease-free water 4. Trans2K DNA Marker

Procedure: Add the materials into new EP tubes in the order of following table respectively.

group 1 2
sample 7 μL pGAL1(608.7 ng/μL) 3 μL pGAL1(608.7 ng/μL)
10×Tango Buffer(μL) 2 1
nuclease-free water(μL) 0 5.3
EcoRI(μL) 0.5 0
PstI(μL) 0.5 0
SpeI(μL) 0 0.5
XbaI(μL) 0 0.2

Mix gently and incubate at 37 degree Celsius for 2.5 hours to group 1 and for 3 hours to group 2.

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of digestion of pGal1 (from left to right:marker,group-1,control-1,group-2,control-2) The picture shows that four kind of enzyme is OK while it has low efficiency.

Recorder: Kaiyue Ma Colony picking

  1. Pick 2 colonies of a plate containing pYeScGAP in 2 test tubes (5 mL LB liquid medium, amp+).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Date: 7.30

Recorder: Yonghao Liang Plasmid Extraction for SUP35 expression plasmids

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O(60°C), 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample 1 2 3 4
A260/A280 1.91 1.87 1.88 1.95
concentration(ng/μL) 50.5 76.9 166.4 50.8

Recorder: Yinchenguang Lyu Plasmid Extraction for YeScGAP expression plasmids

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O(60°C) to one tube, 50 μL WHH into another, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample ddH2O WHH
A260/A280 1.89 1.88
concentration(ng/μL) 192.6 246.5

Recorder: Yinan Chen Plasmid Extraction for GFP expression plasmids

Procedure: 1. Put bacteria solution into 4 EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O(60°C) to two tube, 50 μL WHH into other two, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample ddH2O:1 ddH2O:2 WHH:1 WHH:2
A260/A280 1.92 1.92 1.89 1.89
concentration(ng/μL) 60.5 52.2 69.7 57.1

Recorder: Chengle Zhang DNA concentration measurement with electrophoresis

Material: 1. pGal1 gene in plasmid BBa-J63010(from iGEM kit plate) 2. 10000× gel red 3. Trans2K plus II DNA Marker 4. 6× loading buffer

Procedure: 1. Add 1.0 g agarose to 100 mL 1× TAE buffer. 2. Dissolved by heating. 3. Cool down.Add 2 μL 10000× gel red. 4. Pour into electrophoresis tank. 5. Make up samples as the following table.

(p1 refers to pGal1-1(497.0 ng/μL),p2 refers to pGal1-1(381.8 ng/μL),p2 refers to pGal1-1(368.7 ng/μL),water refers to nuclease-free water,total volume is 10 μL)

p1:water(μL) p2:water(μL) p3:water(μL)
group-1 0.5:9.5 0.5:9.5 0.5:9.5
group-2 1:9 1:9 1:9
group-3 2:8 2:8 2:8
group-4 4:6 4:6 4:6
  1. Add 2 μL 6× loading buffer into each test tube.Then load all samples and 5 μL marker.
  2. Electrophoresis gel：110 V 30 min.
  3. Autoradiography(UV).

Results: 图片名称 (from left to right:marker,p1-1,p1-2,p1-3,p1-4,blank,p2-1,p2-2,p2-3,p2-4,blank,p3-1,p3-2,p3-3,p3-4,p4-1,p4-2,p4-3,p4-4)

It shows that adding gel red to the gel has a striking result.While it will cost much gel red every time.Maybe we can decrease the gel red next time.

Recorder: Xuefeng Meng PCR of sup35

Experimental materials PCR products of sup35, primer 1 & primer 2(10 μM), Sterilized ddH2O, 2×HiFi-PCR Master

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample 1（233.9ng/μL） 2（23.39ng/μL） 3（2.339ng/μL） 4（0.2339ng/μL）
Sterilized ddH2O 20 μL 20 μL 20 μL 20 μL
2×HiFi-PCR Master 25 μL 25 μL 25 μL 25 μL
template 1 μL 1 μL 1 μL 1 μL
Primer 1 2 μL 2 μL 2 μL 2 μL
Primer 2 2 μL 2 μL 2 μL 2 μL
total 50 μL 50 μL 50 μL 50 μL

2.PCR reaction Parameters setting：

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) Stored at 4°C.

3.Agarose gel electrophoresis gel 1. Add 0.35 g agarose to 35 mL TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Mix 50 μL PCR product and 10 μL loading buffer. 6. Loading：DNA marker 5 μL, PCR productrs: 30 μL : sample1,1,2,2,3,3,4,4. 7. Electrophoresis gel：110 V 30 min. 8. Autoradiography(UV).

Result

marker:

Recorder: Xuefeng Meng Gel Extraction of PCR products of sup35

Procedure: 1. Cut off the Gel part containing the target band. Get 4 tubes of digestion product. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of digestion product.

sample 1 2 3 4
concentration(ng/μL) 16.9 13.4 19.8 19.9
A260/A280 1.98 1.97 1.89 1.82
A260/A230 0.06 0.15 0.19 0.03

Recorder：Yonghao Liang **Colony picking of SUP35 **

  1. Pick 4 colonies from 1 plate in 4 test tubes (5 mL LB liquid medium & 5μL kn+).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder: Ya Jiang PCR of pGAl

Experimental materials 1. plasmids containing pGAL1(BBa_J63006) 2. primer VF2 & primer VR(25 μM) 3. Sterilized ddH2O 4. 2×HiFi-PCR Master 5. Trans2K plus II DNA Marker

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample 1（447.0ng/μL） 2（381.8ng/μL） 3（368.7ng/μL）
Sterilized ddH2O 11 μL 11 μL 11 μL
2×HiFi-PCR Master 12.5 μL 12.5 μL 12.5 μL
template 0.5 μL 0.5 μL 0.5 μL
Primer 1 0.5 μL 0.5 μL 0.5 μL
Primer 2 0.5 μL 0.5 μL 0.5 μL
total 25 μL 25 μL 25 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 56 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis gel (100 mL gel contains 2 μL 10000× gel red) Result

Recorder: Yinchenguang Lyu & Yonghao Liang Standardization of sup35

Experimental materials 1. PCR product of sup35. 2. Primer: standard-sup35-p, standard-sup35-r. Designed by ourselves, synthesized by Sangon Biotech; 3. Sterilized ddH2O; 4. PrimeSTAR Max Premix(2X) bought from TaKaRa, Code No. R010A.

About primers

primer 1 2
sequence 5'-CCGGAATTCGCG
GCCGCTTCTAGA
TGTCGGATTCAA
ACCAGGGCAACA-3'		 5'-TGCACTGCAGCG
GCCGCTACTAGT
AATCATTCACCA
CCTCATCGTCAA
CTTC-3'

Procedure:

1.Prepare 2 PCR tubes(each sample is put into 1 tubes)and sequentially add：

sample
Sterilized ddH2O 11 μL
PrimeSTAR Max Premix(2X) 12.5 μL
Template(PCR product 23.39ng/μL;233.9ng/μL) 0.5 μL
standard-sup35-p 0.5 μL
standard-sup35-r 0.5 μL
total 25 μL

2.PCR reaction Parameters setting：

stage temperature time
step 1 95 5 min
step 2 95 10 s
step 3 56 15 s
step 4 72 50 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

Recorder: Chengle Zhang Colony picking

  1. Pick 3 colonies of each plate (containing Yeast constitutive promoter TEF2 and Kozak sequence) in 6 test tubes (5 mL LB liquid medium).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Date: 7.31

Recorder: Yinchenguang Lyu & Yonghao Liang Agarose Gel Electrophoresis of Standardization sup35 PCR Production 图片名称

Recorder: Yinchenguang Lyu Plasmid Extraction for sup35 expression plasmids

Procedure: 1. Put bacteria solution into 8 EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL WHH into the four tubes, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with WHH at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

sample 1 2 3 4
A260/A280 1.87 1.86 1.91 1.90
concentration(ng/μL) 82.6 79.8 51.0 40.7

Recorder: Kaiyue Ma and Chengle Zhang PCR of bacteria containing recombinant plasmid of Kozak and pTEF2

About primers

primer 1 2
sequence 5'- TGCCACCTGACG
TCTAAGAA-3'		 5'- ATTACCGCCTTT
GAGTGAGC-3'

Procedure:

1.Prepare 5 PCR tubes and sequentially add：

sample 1,2,3,4,5
Sterilized ddH2O 16.5 μL
5×PrimeSTAR Buffer (Mg2+Plus) 5 μL
dNTP Mixture (2.5 mM each) 2 μL
Template(bacteria) 0.75 μL
Primer 1 0.5 μL
Primer 2 0.5 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl) 0.25 μL
total 25 μL

2.PCR reaction Parameters setting：

stage temperature time
step 1 95 10 min
step 2 95 10 s
step 3 55 15 s
step 4 72 47 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product (from left to right: marker,sample 1,2,3,4,5)

Recorder: Xuefeng Meng PCR of sup35

Experimental materials PCR products of sup35, primer 1 & primer 2(10 μM), Sterilized ddH2O, 2×HiFi-PCR Master

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample 1（82.6 ng/μL） 2（8.26 ng/μL） 3（79.8 ng/μL） 4（79.8 ng/μL）
Sterilized ddH2O 22 μL 22 μL 20 μL 22 μL
2×HiFi-PCR Master 25 μL 25 μL 25 μL 25 μL
template 1 μL 1 μL 1 μL 1 μL
Primer 1 1 μL 1 μL 2 μL 1 μL
Primer 2 1 μL 1 μL 2 μL 1 μL
total 50 μL 50 μL 50 μL 50 μL

2.PCR reaction Parameters setting：

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

35 cycles(step 2 ~ step 4) Stored at 4°C.

3.Agarose gel electrophoresis gel 1. Add 0.35 g agarose to 35 mL TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Mix 50 μL PCR product and 10 μL loading buffer. 6. Loading：DNA marker 5 μL, PCR productrs: 30 μL : sample1,1,2,2,3,3,4,4. 7. Electrophoresis gel：110 V 30 min. 8. Autoradiography(UV).

Result

marker:

Date: 8.1

Recorder: Chengle Zhang & Yu Xie Colony picking of Kozak

  1. Pick 10 colonies of a plate containing Kozak in 10 test tubes (5 mL LB liquid medium, Cm+).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder：Yin Wu Double Digestion of sfGFP1-10 and KOZAK

Procedure: Add the materials into new EP tubes in the order of following table respectively.

sample 1 2 3 4
nuclease free water(μL) 14 14 14 14
10× Buffer(μL) 4 4 4 4
plasmid of KOZAK 0 0 20 20
sfGFP1-10 20 20 0 0
PstI(μL) 1 1 1 1
XbaI(μL) 1 1 0 0
BcuI(μL) 0 0 1 1
total(μL) 40 40 40 40

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

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of digestion of sfGFP1-10 and KOZAK (from left to right:marker, sample 1-8)

Recorder: Kaiyue Ma Double Digestion of pET28a plasmid containing sup35

Procedure: Add the materials into new PCR tubes in the order of following table respectively.

sample 1 2
10× Green Buffer(μL) 2 2
plasmid 17 17
EcoRI(μL) 0.5 0.5
XbaI(μL) 0.5 0.5
total(μL) 20 20

Mix gently and incubate at 37 degree Celsius The thermal cycler. 30 minutes for tube 1, and 1 hour for tube 2.

Recorder: Yin Wu, Ya Jiang Gel Extraction of digestion product of sfGFP and KOZAK

Procedure: 1. Cut off the Gel part containing the target band. Get 8 tubes of digestion product. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of digestion product.

sample sfGFP1-10-1 sfGFP1-10-2 KOZAK-1 KOZAK-2
A260/A280 3.3 5.8 26.2 11.2
concentration(ng/μL) 2.30 1.76 1.93 2.28

Recorder: Yu Xie Colony picking of PR+GFP （13:10）

  1. Pick 2 colonies of a plate containing PR+GFP in 2 test tubes (5 mL LB liquid medium, kana).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min.

Recorder: Chengle Zhang PCR of AD and BD

Experimental materials 1. Plasmid: pGAD-T7 and pGBK-T7(5 μL for each) from laboratory 436 of USTCSLS, Mr Liao; 2. Primer: AD 1, AD 2, BD 1, BD 2. Designed by ourselves, synthesized by Sheng Gong; 3. Sterilized ddH2O, 2×HiFi-PCR Master, bought from Sheng Gong.

About primers

primer AD 1 AD 2 BD 1 BD 2
sequence 5'-CCGGAATTCGC
GGCCGCTTCTAG
ATGGCCAATTTT
AATCAAAGTGGG
AATATTGCTG-3'		 5'-AACTGCAGCGG
CCGCTACTAGTA
CTACTCTTTTTT
TGGGTTTGGTGG
GGTATC-3'		 5'-CCGGAATTCGC
GGCCGCTTCTAG
ATGAAGCTACTG
TCTTCTATCGAA
C-3'		 5'-AACTGCAGCGG
CCGCTACTAGTA
CTACGATACAGT
CAACTGTCTTTG
ACC-3'

Procedure:

1.Prepare 2 PCR tubes and sequentially add：

sample AD BD
Sterilized ddH2O 22 μL 22 μL
2×HiFi-PCR Master 25 μL 25 μL
pGAD-T7 (542.5 μg/mL) 1 μL 0
pGBK-T7 (506.7 μg/mL) 0 1 μL
AD Primer-1(10 μM) 2 μL 0
AD Primer-2(10 μM) 2 μL 0
BD Primer-1(10 μM) 0 2 μL
BD Primer-2(10 μM) 0 2 μL
total 50 μL 50 μL

2.PCR reaction Parameters setting：

stage temperature time
step 1 95 10 min
step 2 95 10 s
step 3 55 15 s
step 4 72 45 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

3.Agarose gel electrophoresis Result:

Recorder:Yinchenguang Lyu, Chengle Zhang Result of Gel Extraction of the product of last step |sample|AD|BD| |-| |concentration(ng/μL)|66.6|7.5| |260/280|1.97|3.48| |260/230|0.09|0.01|

Recorder: Yin Wu Ligation of sfGFP1-10 and KOZAK

Material: 1. double digestion product of sfGFP1-10 2. double digestion product of KOZAK 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: Add to the sample: 15 μL sfGFP 3 μL KOZAK 2 μL 10* T4 DNA Ligase Buffer 0.25 μL T4 DNA Ligase

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

Recorder: Yin Wu Transformation of recombinant plasmid of KOZAK and sfGFP1-10

Materials(all made by ourselves): 1. recombinant plasmid of KOZAK and sfGFP1-10 2. SOC media 3. competent cells

Procedure: 1. Put competent cells on ice. Then pre-chill by placing the tubes on ice. 2. Pipet the recombinant plasmid into competent cell tubes, 10 µL for each. 3. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. 4. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. 5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. 6. Add 200 µL of SOC media per tube, and incubate at 37°C for 1 hours. 7. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. 8. Discard 150 μL supernatant liquid and resuspend the bacteria. 9. Coat plate: add the solution to 2 plates (Cm+) and spread it, respectively, then cultivate it overnight.

Recorder: Ya Jiang Colony picking

  1. Pick 4 colonies of a plate containing sfGFP in 4 test tubes (5 mL LB liquid medium, Cm+).
  2. Cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Recorder: Yinchenguang Lyu, Yonghao Liang Standardization of sup35

Experimental materials 1. PCR product of sup35, plasmid containing sup35. 2. Primer: standard-sup35-p, standard-sup35-r. Designed by ourselves, synthesized by Sangon Biotech; 3. Sterilized ddH2O; 4.2×HiFi-PCR Master.

About primers

primer 1 2
sequence 5'-CCGGAATTCGCG
GCCGCTTCTAGA
TGTCGGATTCAA
ACCAGGGCAACA-3'		 5'-TGCACTGCAGCG
GCCGCTACTAGT
AATCATTCACCA
CCTCATCGTCAA
CTTC-3'

Procedure:

1.Prepare 4 PCR tubes and two contianed sample 1, the other two contained sample 2：

sample 1 (plasmid 51.0ng/μL) 2 (plasmid 79.8ng/μL)
Sterilized ddH2O 22.0 μL 22.0 μL
Template 1 μL 1 μL
standard-sup35-p 1 μL 1 μL
standard-sup35-r 1 μL 1 μL
2×HiFi-PCR Master 25 μL 25 μL
total 50 μL 50 μL

2.PCR reaction Parameters setting：

stage temperature time
step 1 95 5 min
step 2 95 1 min
step 3 57 1 min
step 4 72 1 min50 s
step 5 72 10 min
step 6 4 --

35 cycles(step 2 ~ step 4)

results failed. sample: from left to right: marker, sample 2,2(50 μL PCR product+10 μL loading buffer)

Plasmid Extraction for plasmids containing Kozak(BBa_K165002) Recorder: Yonghao Liang ,Chenguang Lyu and Yu Xie

Procedure: 1. Put bacteria solution into two EP tube respectively. Centrifuge bacteria solution at 11000 rpm, few sediment got. Remove the supernatant. Repeat once. 2. Add 250 μL Buffer P1, suspend cells. 3. Add 250 μL Buffer P2, mix well, 3 min's standing. 4. Add 350 μL Buffer P3, mix well. 5. 13400 rpm centrifuge 10 min, move all supernatant to adsorption column, 11000 rpm centrifuge 30 s, discard filtrate. Repeat until all the supernatant from the two tube is adsorped. 6. Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate. 7. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 8. 12000 rpm centifuge 1 min. 9. Lying for 10 min. 10. Put the adsorption column in a new EP tube. Add 50 μL ddH2O, 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH2O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the density of plasmids.

The OD A260/A280,OD A260/A280 and the concentration of plasmids are as follows:

sample Kozak-1 Kozak-2 Kozak-3 Kozak-4 Kozak-5 Kozak-6 Kozak-7 Kozak-8 Kozak-9 Kozak-10
A260/A280 1.92 1.81 1.77 1.92 1.92 1.74 1.92 2.07 1.83 1.94
A260/A230 2.05 1.31 1.15 2.03 1.09 0.95 1.52 1.28 0.83 0.34
concentration(ng/L) 41.5 62.6 60.4 38.8 17.5 34.7 29.3 19.6 24.6 18.4

Then we check the plasmid with fastdigest enzyme EcoRI and PstI.

Agarose gel electrophoresis Result:

(from left to right: Kozak-1,Kozak-2,Kozak-3,Kozak-4,Kozak-5,Kozak-6,Kozak-7,Kozak-8,Kozak-9,Kozak-10)

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