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Revision as of 20:28, 6 October 2018
Wet Lab
July 1
Cloning
- Received and resuspended of the IDT basic part
- Sample
- Enterocin A
- Enterocin B
- Enterocin 96
- Bovicin HJ50
- Duracin TW49M
- Lacticin Z
- Leucocyclicin Q
- Subtilosin
July 2
None
July 3
Growth curve exp
- Observe the growth population of Bacillis subtilis in different temperature Condition: 37°C, 32°C, 27°C
July 4
None
July 5
None
July 6
None
July 7
None
July 8
Growth curve exp
- Observe the growth population of Bacillis subtilis in different pH Condition: pH = 4, 5, 6, 7, 8
July 9
None
July 10
None
July 11
None
July 12
None
July 13
None
July 14
None
July 15
Expression
- repared competent cell E. coli ER2566
Growth curve exp
- Observe the growth population of Bacillis subtilis in different salinity Condition: 0.17M, 0.25M, 0.5M, 0.75M, 1.0M
- Sample
July 16
Cloning
- Amplify the insert gene (Pfu PCR)
- Electrophoresis (To check PCR products)
- Sample
- Lacticin Z + pTXB1
- Bovincin HJ50+ pTXB1
July 17
Cloning
- Digestion of PCR products (Including insert and backbone pTXB1)
- Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
- Sample
- Lacticin Z + pTXB1
- Bovincin HJ50+ pTXB1
July 18
Cloning
- Transformation of ligation products (Transform into E. coli DH5α)
- Taq PCR (PCR of ligation products to amplify insert gene)
- Electrophoresis (To check PCR products)
- Sample
- Lacticin Z + pTXB1
- Bovincin HJ50+ pTXB1
July 19
Cloning
- Cultivation
- Miniprep (Purify Plasmid)
- Sample
- Lacticin Z + pTXB1
- Bovincin HJ50+ pTXB1
July 20
Cloning
- PurposeL: Sequencing plasmid
- Sample
- Lacticin Z + pTXB1
- Bovincin HJ50+ pTXB1
July 21
Cultivation
July 22
Cloning
- Amplify the insert gene (Pfu PCR)
- Electrophoresis (To check PCR products)
- Digestion of PCR products (Including insert and backbone pTXB1)
- Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
- Sample
- Leucocyclicin Q + pTXB1
July 23
Cloning
- Transformation of ligation products (Transform into E. coli DH5α)
- Taq PCR (PCR of ligation products to amplify insert gene)
- Electrophoresis (To check PCR products)
- Sample
- Leucocyclicin Q + pTXB1
July 24
Cloning
- Cultivation
- Miniprep (Purify Plasmid)
- Sample
- Leucocyclicin Q + pTXB1
July 25
Cloning
- Sequencing plasmid
- Sample
- Leucocyclicin Q + pTXB1
July 26
None
July 27
None
July 28
None
July 29
Expression
- Transformation (Transform correct plasmid into E. coli ER2566)
- Leucocyclicin Q + pTXB1
July 30
None
July 31
None
August 1
None
August 2
None
August 3
None
August 4
None
August 5
Expression
- Purepose: Transformation (Transform correct plasmid into E. coli ER2566)
- sample
- Leucocyclicin Q + pTXB1
August 6
Expression
- Purepose: Cultivation (Cultivation of E. coli ER2566 colonies for IPTG induction)
- Purepose: IPTG Induction (To test the OD levels of E. coli ER2566 to induce)Condition: OD = 0.5, 1.3
- sample
- Leucocyclicin Q + pTXB1
August 7
Expression
- Purepose: SDS PAGE (To check the protein production after induction)
- sample
- Leucocyclicin Q + pTXB1
August 8
None
August 9
None
August 10
None
August 11
Cloning
- Purepose: Amplify the insert gene (Pfu PCR)
- Purepose: Electrophoresis (To check PCR products)
- sample
- Enterocin A
- Enterocin B
- Enterocin 96
- Duracin TW49M
- Subtilosin
August 12
Cloning
- Purepose: Digestion of PCR products (Including insert and backbone pTXB1)
- Purepose: Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
- Purepose: Transformation of ligation products (Transform into E. coli DH5α)
- sample
- Enterocin A + pTXB1
- Enterocin B + pTXB1
- Enterocin 96 + pTXB1
- Duracin TW49M + pTXB1
- Subtilosin + pTXB1
Growth curve
- Purepose: Observe the growth of Bacillus subtilis in different temperature, pH, salinityCondition:
- 1.Temp: 37°C , pH:7 , salinity: 0.17M
- 2.Temp: 30°C , pH:9 , salinity: 0.5M
- 3.Temp: 25°C , pH:5 , salinity: 0.25M
August 13
Cloning
- Purepose: Taq PCR (PCR of ligation products to amplify insert gene)
- Purepose: Electrophoresis (To check PCR products)
- sample
- Enterocin A + pTXB1
- Enterocin B + pTXB1
- Enterocin 96 + pTXB1
- Duracin TW49M + pTXB1
- Subtilosin + pTXB1
August 14
Cloning
- Cultivation
- Miniprep (Purify Plasmid)
- Sample
- Enterocin A + pTXB1
- Enterocin B + pTXB1
- Enterocin 96 + pTXB1
- Duracin TW49M + pTXB1
- Subtilosin + pTXB1
Expression
- Transformation (Transform correct plasmid into E. coli ER2566)to test different cultivation temperature
August 15
Cloning
- Purepose: Sequencing plasmid
- Sample
- Enterocin A + pTXB1
- Enterocin B + pTXB1
- Enterocin 96 + pTXB1
- Duracin TW49M + pTXB1
- Subtilosin + pTXB1
Expression
- Purepose: Cultivation (Cultivation of E. coli ER2566 colonies for IPTG induction)
- Purepose: IPTG Induction (To test the temperature of cultivation after induction)Condition: temp = 37, 30, 13.5°C
August 16
Expression
- Purepose: SDS PAGE (To check the protein production after induction)Check different temperature
- Purepose: IPTG Induction (To test the concentration of IPTG)Condition: concentration = 200, 400, 600, 800, 1000μM
August 17
Expression
- Purepose: SDS PAGE (To check the protein production after induction)Check different IPTG concentration
- Sample
August 18
SDS-PAGE
August 19
Cloning
- Purepose: Digestion of PCR products (Including insert and backbone pET30a)
- Purepose: Ligation of digestion products (Ligase the DNA fragment into backbone pET30a)
- Sample(8/17)
- Enterocin A + pET30a
- Enterocin B + pET30a
- Enterocin 96 + pET30a
- Bovicin HJ50 + pET30a
- Duracin TW49M + pET30a
- Lacticin Z + pET30a
- Leucocyclicin Q + pET30a
- Subtilosin + pET30a
August 20
Cloning
- Purepose: Transformation of ligation products (Transform into E. coli DH5α)
- Purepose: Taq PCR (PCR of ligation products to amplify insert gene)
- Purepose: Electrophoresis (To check PCR products)
- Sample
- Enterocin A + pET30a
- Enterocin B + pET30a
- Enterocin 96 + pET30a
- Bovicin HJ50 + pET30a
- Duracin TW49M + pET30a
- Lacticin Z + pET30a
- Leucocyclicin Q + pET30a
- Subtilosin + pET30a
August 21
Cloning
- Purepose: Cultivation
- Purepose: Miniprep (Purify Plasmid)
- Sample
- Enterocin A + pET30a
- Enterocin B + pET30a
- Enterocin 96 + pET30a
- Bovicin HJ50 + pET30a
- Duracin TW49M + pET30a
- Lacticin Z + pET30a
- Leucocyclicin Q + pET30a
- Subtilosin + pET30a
August 22
Cloning
- Sequencing plasmid
- Sample
- Enterocin A + pET30a
- Enterocin B + pET30a
- Enterocin 96 + pET30a
- Bovicin HJ50 + pET30a
- Duracin TW49M + pET30a
- Lacticin Z + pET30a
- Leucocyclicin Q + pET30a
- Subtilosin + pET30a
August 23
Protein purification(8/22-8/23)
- Sample
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
Cultivation and IPTG induction
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar 8/20
- Sf1a-lectin-his Ar 8/20
- Add 1000μM IPTG and test OD600
Sonication
- Sample
- Hv1a-his Ar 8/23
- Sf1a-lectin-his Ar 8/23
August 24
Protein purification(8/23-8/24)
- Sample
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
Cultivation and IPTG induction
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Sf1a-his Ar 8/20
- Sf1a-lectin-his Ar 8/20
- Refresh to approximately OD600 0.7
- Add 1000μM IPTG and test OD600
- Sf1a-his: 0.966
- Sf1a-lectin-his: 1.363
- Certification, and add 2mL binding buffer and 200μM PMSF for sonication. Repeat three times.
Sonication
- Sample
- Sf1a-his Ar 8/24
- Sf1a-lectin-his Ar 8/24
SDS-PAGE
- Check the purification protein.
- Sample
- Hv1a-lectin-his(8/22-8/23)
August 25
Protein purification(8/24-8/25)
- Sample
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample
- Hv1a-lectin-his(8/23-8/24)
August 26
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar 8/15
- Hv1a-lectin-his Ar 8/15
- Sf1a-lectin-his Ar 8/15
- OAIP-lectin-his Ar 8/15
Sonication
- Sample
- Sf1a-his Ar 8/24
- Sf1a-lectin-his Ar 8/24
Protein purification
- Sample
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Sf1a-his Ar 8/17
- OAIP-his Ar 8/17
IPTG induction
- Induce the mass protein expression for SDS-PAGE.
- The protein would form inclusion body. Therefore, it was devided into two parts, the urea and control.
- Sample
- Hv1a-his Ar 8/26
- Hv1a-lectin-his Ar 8/26
- Sf1a-lectin-his Ar 8/26
- OAIP-lectin-his Ar 8/26
- Add 500μM IPTG
- Take the following samples to go on the experience first. The others storage in 4℃ refrigerator.
- Hv1a-his Ar 8/26
- Hv1a-lectin-his Ar 8/26
SDS-PAGE
- Check the protein in pellet or in solution.
- Sample
- Hv1a-his Ar 8/26(with urea)
- Hv1a-his Ar 8/26
- Hv1a-lectin-his Ar 8/26(with urea)
- Hv1a-lectin-his Ar 8/26
- Sonication and the supernatant is loading sample1
- 10 seconds on, 40 seconds off and repeat six times.
- Add binding buffer (with/without urea) again and the solution is loading sample 2
- Sonication again and the supernatant is loading sample 3
- 10 seconds on, 40 seconds off and repeat six times.
- Add binding buffer (with/without urea) again and the solution is loading sample4
- Loading sample
- Hv1a-his (with urea) 1~4
- Hv1a-his 1~4
- Hv1a-lectin-his (with urea) 1~4
- Hv1a-lectin-his 1~4
IPTG induction
- Induce the mass protein expression for SDS-PAGE.
- The protein would form inclusion body. Therefore, it was devided into two parts, the urea and control.
- Sample
- Hv1a-his Ar 8/26
- Hv1a-lectin-his Ar 8/26
- Sf1a-lectin-his Ar 8/26
- OAIP-lectin-his Ar 8/26
- Add 500μM IPTG and test OD600 to approximately 1.050
SDS-PAGE
- Check the protein in pellet or in solution.
- Sample
- Hv1a-his Ar 8/26(with urea)
- Hv1a-his Ar 8/26
- Hv1a-lectin-his Ar 8/26(with urea)
- Hv1a-lectin-his Ar 8/26
- Sf1a-lectin-his Ar 8/26(with urea)
- Sf1a-lectin-his Ar 8/26
- OAIPlectin-his Ar 8/26(with urea)
- OAIP-lectin-his Ar 8/26
- Certificate the sample, add binding buffer (with/without urea), PMSF
- Sonication and the supernatant is loading sample1
- 10 seconds on, 40 seconds off and repeat six times.
- Add binding buffer (with/without urea) again and the solution is loading sample 2
- Sonication again and the supernatant is loading sample 3
- 10 seconds on, 40 seconds off and repeat six times.
- Add binding buffer (with/without urea) again and the solution is loading sample4
- Loading sample
- Hv1a-his (with urea) 1~4
- Hv1a-his 1~4
- Hv1a-lectin-his (with urea) 1~4
- Hv1a-lectin-his 1~4
- Sf1a-lectin-his (with urea) 1~4
- Sf1a-lectin-his 1~4
- OAIPlectin-his (with urea) 1~4
- OAIP-lectin-his 1~4
August 27
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
August 28
Protein purification
- The buffer contains the urea
- Sample(8/27)
- Hv1a-his
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample(8/28)
- Hv1a-his
- Hv1a-lectin-his
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Sf1a-his Ar 8/17
August 29
Protein purification
- The buffer contains the urea
- Sample(8/28)
- Sf1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample(8/29)
- Sf1a-lectin-his
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Sf1a-his Ar 8/17
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar 8/15
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-lectin-his Ar 8/15
- Sf1a-lectin-his Ar 8/15
- OAIP-lectin-his Ar 8/15
August 30
IPTG induction
- Induce the mass protein expression for SDS-PAGE.
- The protein would form inclusion body. Therefore, it was devided into two parts, the urea and control.
- Sample
- Hv1a-lectin-his Ar 8/29
- Sf1a-lectin-his Ar 8/29
- OAIP-lectin-his Ar 8/29
- Refresh to approximately OD600 0.6
- Add 500μM IPTG and cultivate for 4hr
Protein purification
- The buffer contains the urea
- Sample(8/30)
- Sf1a-lectin-his
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample(8/29)
- Sf1a-lectin-his
- Hv1a-lectin-his
PCR
- amplify the insert gene
- Sample(8/30)
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
Electrophoresis
- Electrophoresis of PCR product
- Sample(8/30 PCR product)
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
Digestion
- Digest min for transformation
- Sample
- Hv1a-his digestion EP
- Hv1a-lectin-his digestion ES
- Sf1a-his digestion EP
- Sf1a-lectin-his digestion ES
- OAIP-his digestion EP
- OAIP-lectin-his digestion ES
Transformation
- Transformation PSB1C3 to DH5α
- Sample
- Trans PSB1C3 8/30
- Trans PSB1C3 8/30
Urea test
- Check whether protein in inclusion body or not.
- Sample
- Hv1a-his
- Hv1a-his + Urea
- Sf1a-his
- Sf1a-his + Urea
- OAIP-his
- OAIP-his + Urea
August 31
Ligation
- Ligase insert gene into the pSB1C3
- Sample
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
Transformation
- Transformation of ligated product to DH5α
- Sample(8/31 Ligation product)
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
Dialysis and refolding
- Dialysis and refolding for insoluble protein
- Sample(8/8)
- Hv1a-lectin-his (8/30)
- Sf1a-lectin-his(8/30)
- OAIP-lectin-his(8/30)
- Hv1a-his(8/28)
SDS-PAGE
- Check the purification protein.
- Sample(8/30)
- Sf1a-lectin-his
- Hv1a-lectin-his
September 1
Protein purification
- The buffer contains the urea
- Sample(8/30)
- OAIP-lectin-his
- Hv1a-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample(8/30)
- OAIP-lectin-his
- Hv1a-his
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- OAIP-his Ar
- Sf1a-his Ar
IPTG Induction
- Prepare for the purify the protein from sample BL21-Rosetta-gami colonies.
- Sample
- Hv1a-his Ar
- OAIP-his Ar
- Sf1a-his Ar
- Add IPTG 500μM and test OD600 after 4hr.
Sonication
- Sample
- Hv1a-his Ar
- OAIP-his Ar
- Sf1a-his Ar
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
Sonication
- Sample
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
September 2
Protein purification
- The buffer contains the urea
- Sample
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
Protein quantification Braford method
- Sample
- Hv1a-his
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
Dialysis and refolding
- Dialysis and refolding for insoluble protein
- Sample
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
September 3
Protein purification
- Sample
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
Protein quantification Braford method
- The buffer contains the urea
- Sample
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
Concentrate protein
- Increase the concentration of protein
- Sample
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
September 4
Protein purification
- The buffer contains the urea
- Sample
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
September 5
IPTG Induction
- Prepare for the purify the protein from sample BL21-Rosetta-gami colonies.
- Sample
- Hv1a-his
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
- Add IPTG 500μM and test OD600 after 4hr.
Sonication
- Sample
- Hv1a-his
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
Protein purification
- The buffer contains the urea
- Sample
- Hv1a-his
- Hv1a-lectin-his
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
UV test
- Test degradation rate
- Sample
- Hv1a-his
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
Concentrate protein
- Increase the concentration of protein
- Sample
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
Protein quantification Braford method
- Sample
- Hv1a-lectin-his Ar
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
September 6
Protein purification
- The buffer contains the urea
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
Protein quantification Braford method
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 7
Protein purification
- The buffer contains the urea
- Sample
- Sf1a-lectin-his Ar
- OAIP-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample
- OAIP-lectin-his Ar
- Sf1a-lectin-his Ar
UV test
- Test degradation rate
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
Sonication
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
Protein quantification Braford method
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 8
Protein purification
- The buffer contains the urea
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
Cultivation and IPTG induction
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
- Refresh to approximately OD600 0.7
- Add 1000μM IPTG and test OD600
- Hv1a-his:0.772
- Hv1a-lectin-his:0.631
- Sf1a-his:0.567
- Sf1a-lectin-his:0.78
- OAIP-his:0.851
- OAIP-lectin-his:0.819
- Certification, and add 2mL binding buffer and 200μM PMSF for sonication. Repeat three times.
Sonication
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
Sonication
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
Protein purification
- The buffer contains the urea
- Sample
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 9
Protein purification
- The buffer contains the urea
- Sample
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
- Collect：
pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH2O solution.
SDS-PAGE
- Check the purification protein.
- Sample
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 10
None
September 11
Dialysis protein
- Dialysis and refolding for insoluble protein
- Sample
- Hv1a-his E1-E3
- Hv1a-lectin-his E1-E5
- Sf1a-his E1-E5
- Sf1a-lectin-his E1-E4
- OAIP-his E1-E3
- OAIP-lectin-his E1-E2
September 12
Protein quantification Braford method
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 13
None
September 14
None
September 15
None
September 16
None
September 17
Dialysis protein
- Dialysis and refolding for insoluble protein
- Sample
- Hv1a-his E2-E3
- Hv1a-lectin-his E1-E6
- Sf1a-his E1-E4
- Sf1a-lectin-his E2-E4
- OAIP-his E2-E4
- OAIP-lectin-his E2-E3
September 18
None
September 19
None
September 20
None
September 21
None
September 22
Protein quantification Braford method
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 23
Dialysis protein
- Remove urea
- Sample
- Hv1a-his E1-E3
- Hv1a-lectin-his E1-E6
- Sf1a-his E1-E4
- Sf1a-lectin-his E1-E6
- OAIP-his E1-E3
- OAIP-lectin-his E1-E6
SDS-PAGE
- Check the purification protein again
- Sample
- Hv1a-his
- Hv1a-lectin-his (no β-me)
- Sf1a-his
- Sf1a-lectin-his (no β-me)
- OAIP-his
- OAIP-lectin-his (no β-me)
Electrophoresis
- Electrophoresis of composite parts again
- Sample(8/30 PCR product)
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
September 24
Electrophoresis
- Electrophoresis of composite parts again
- Sample(8/30 PCR product)
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
September 25
Protein quantification Braford method
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- Sf1a-lectin-his Ar
- OAIP-his Ar
- OAIP-lectin-his Ar
September 26
None
September 27
None
September 28
Protein quantification Braford method
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- Sf1a-his Ar
- OAIP-his Ar
September 29
None
September 30
None
October-1
None
October 2
None
October 3
None
October 4
None
October 5
Cultivation
- For larva test
- Sample
- Hv1a-GS linker-lectin Ar
October 6
None
October 7
None
October 8
Protein quantification Braford method
- Sample
- HLG-1
- HLG-2
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
- BL
- RG
Protein quantification Braford method
- Sample
- HLG-1
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
October 9
None
October 10
None
October 11
Protein quantification Braford method
- Sample
- HLG-1
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
- RG
SDS-PAGE
- Check the purification protein.
- Sample
- Hv1a(sonication)
- Hv1a(purification)
- Sf1a(sonication)
- Sf1a(purification)
- OAIP(sonication)
- OAIP(purification)
Protein quantification Braford method
- Sample
- HLG
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
Protein quantification Braford method
- Sample
- HLG(purification)
- Hv1a-his(purification)
- Hv1a-lectin-his(purification)
- Sf1a-his(purification)
- Sf1a-lectin-his(purification)
- OAIP-his(purification)
- OAIP-lectin-his(purification)
- Hv1a-his(sonication)
- OAIP-his(sonication)
- OAIP-lectin-his(sonication)
- HGL-his(sonication)
- Hv1a-his
- OAIP-lectin-his
October 12
None
October 13
None
October 14
None
October 15
SDS-PAGE
- Check the purification protein.
- Sample
- Hv1a-his
- Hv1a-lectin-his
- Sf1a-his
- Sf1a-lectin-his
- OAIP-his
- OAIP-lectin-his
October 16
None
October 17
None
October 18
None
October 19
None
