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Revision as of 08:10, 23 September 2018
Wet Lab
July 1
None
July 2
None
July 3
none
July 4
None
July 5
Restore IDT in gblock fragment
- Save DNA
- Sample
- Hv1a-his-Sf1a NO.98351612
- Sf1a-his- OAIP NO.98351613
- OAIP-his- Hv1a NO.98351614
- Hv1a-lectin-his NO.98351615
- Sf1a-lectin-his NO.98351616
- OAIP-lectin-his NO.98351617
July 6
None
July 7
None
July 8
None
July 9
PCR of IDT gblock fragment
- The IDT PCR amplify the insert gene and use phusion DNA polymerase (PFU) to improve the PCR accuracy rate.
- Sample
- Hv1a-his-Sf1a
- Sf1a-his- OAIP
- OAIP-his- Hv1a
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Electrophoresis
- Check IDT PCR product
- Sample(7/9 PCR product)
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
July 10
PCR of IDT gblock fragment
- Amplify the DNA to revise the mistake.
- Sample
- Hv1a-his-Sf1a
- Sf1a-his- OAIP
- OAIP-his- Hv1a
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Electrophoresis
- Check IDT PCR product
- Sample(7/10 PCR product)
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Gel extraction
- For the purification of DNA, we cut the correct electrophoresis band to do gel extraction.
- Sample
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Digestion
- Digest the gene fragment(7/10) and pSB1C3 with EcoRI and PstI to ligase the gene fragment into the pSB1C3.
- Sample
- Hv1a-lectin-his 4 7.10 (EP)
- Sf1a-his 1 7.10 (EP)
- OAIP-his 2 7.10 (EP)
- OAIP-lectin-his 5 7.10 (EP)
- Hv1a-his 3 7.10 (EP)
- Sf1a-lectin-his 6 7.10 (EP)
- pSB1C3 (EP)
Electrophoresis
- Electrophoresis of digested product to check the pSB1C3 digestion.
- Sample
- pSB1C3 (EP)
Ligation
- Ligase DNA fragment into the pSB1C3
- Sample digestion product
- Sample
- Hv1a-lectin-his 4 7.10 (EP)
- Sf1a-his 1 7.10 (EP)
- OAIP-his 2 7.10 (EP)
- OAIP-lectin-his 5 7.10 (EP)
- Hv1a-his 3 7.10 (EP)
- Sf1a-lectin-his 6 7.10 (EP)
- pSB1C3 (EP)
July 11
Transformation:
- Transformation of ligated product to DH5α:
- Sample
- Sf1a-his 1 7.10 Cr
- Hv1a-lectin-his 4 7.10 Cr
- OAIP-his 2 7.10 Cr
- OAIP-lectin-his 5 7.10 Cr
- Hv1a-his 3 7.10 Cr
- Sf1a-lectin-his 6 7.10 Cr
July 12
Electrophoresis
- Electrophoresis of gel extraction product to check the concentration of gene to adjust the ligation protocol.
- Sample
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Transformation:
- Transformation of ligated product 20µL to BL21 competent cell 50µL
- Sample
- Sf1a-his 1 7.10 Cr
- Hv1a-lectin-his 4 7.10 Cr
- OAIP-his 2 7.10 Cr
- OAIP-lectin-his 5 7.10 Cr
- Hv1a-his 3 7.10 Cr
- Sf1a-lectin-his 6 7.10 Cr
July 13
PCR
- PCR of ligated products to amplify the insert gene.
- Sample
- Sf1a-his Cr 1~8
- Hv1a-lectin-his Cr 1~8
- OAIP-his Cr 1~8
- OAIP-lectin-his Cr 1~8
- Hv1a-his Cr 1~8
- Sf1a-lectin-his Cr 1~8
July 14
PCR
- PCR of ligated products to amplify the insert gene.
- Sample
- Sf1a-his Cr 1~4
- Hv1a-lectin-his Cr 1~4
- OAIP-his Cr 1~4
- OAIP-lectin-his Cr 1~4
- Hv1a-his Cr 1~4
- Sf1a-lectin-his Cr 1~4
Electrophoresis
- Electrophoresis of PCR products to check the insert gene base pair
- Sample
- Hv1a-his Cr 1~4
- Sf1a-his Cr 1~4
- OAIP-his Cr 1~4
- Hv1a-lectin-his Cr 1~4
- Sf1a-lectin-his Cr 1~4
- OAIP-lectin-his Cr 1~4
Cultivation
- Cultivation of ligated product.
- Sample
- Sf1a-his Cr 3
- Hv1a-lectin-his Cr 4
- OAIP-his Cr 1
- OAIP-lectin-his Cr 3
- Hv1a-his Cr 3
- Sf1a-lectin-his Cr 4
July 15
PCR
- PCR of ligated products to amplify the insert gene.
- Sample
- Sf1a-his Cr 1~8
- Hv1a-lectin-his Cr 1~8
- Hv1a-his Cr 1~6
- Sf1a-lectin-his Cr 1~3
Electrophoresis
- Electrophoresis of PCR products to check the insert gene base pair
- Sample
- Sf1a-his Cr 1~8
- Hv1a-lectin-his Cr 1~8
- Hv1a-his Cr 1~6
- Sf1a-lectin-his Cr 1~3
Transformation
- Transformation of Hv1a-lectin-his Cr, Hv1a-his Cr into BL21 again.
Miniprep
- Purify plasmid which have insert gene.(7/14)
- Sample
- Hv1a-his Cr
- Sf1a-his Cr
- OAIP-his Cr
- Hv1a-lectin-his Cr
- Sf1a-lectin-his Cr
- OAIP-lectin-his Cr
July 16
Digestion
- Digest the plasmid (7/15) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Sf1a-his 7.15 (EP)
- OAIP-his 7.15 (EP)
- Hv1a-his 7.15 (EP)
- Sf1a-lectin-his 7.15 (EP)
- OAIP-lectin-his 7.15 (EP)
- Hv1a-lectin-his 7.15 (EP)
Electrophoresis
- Electrophoresis of digested product to check the band whether proper to the really base pair.
- Sample(7/16 Digestion product)
- Sf1a-his 7.15 (EP)
- OAIP-his 7.15 (EP)
- Hv1a-his 7.15 (EP)
- Sf1a-lectin-his 7.15 (EP)
- OAIP-lectin-his 7.15 (EP)
- Hv1a-lectin-his 7.15 (EP)
PCR
- PCR of ligated products to amplify the insert gene.
- Sample
- Hv1a-his Cr 1~11
- Hv1a-lectin-his Cr 1~7
Electrophoresis
- Electrophoresis of PCR products to check the insert gene base pair.
- Sample
- Hv1a-his Cr 1~11
- Hv1a-lectin-his Cr 1~7
Cultivation
- Cultivation of ligated product.
- Sample
- Sf1a-his Cr 1
- Hv1a-his Cr 6
- Hv1a-lectin-his Cr 1
- Hv1a-lectin-his Cr 8
- Sf1a-lectin-his Cr 1
July 17
Digestion
- Digest the plasmid (7/15) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Sf1a-his 7.15 (EP)
- Hv1a-his 7.15 (EP)
- Sf1a-lectin-his 7.15 (EP)
- Hv1a-lectin-his 7.15 (EP)
Electrophoresis
- Electrophoresis of digested product to check the band whether proper to the really base pair.
- Sample
- Sf1a-his 7.15 (EP)
- Hv1a-his 7.15 (EP)
- Sf1a-lectin-his 7.15 (EP)
- Hv1a-lectin-his 7.15 (EP)
Miniprep
- Purify plasmid which have insert gene.(7/16)
- Sample
- Sf1a-his Cr 1
- Sf1a-lectin-his Cr 1
- Hv1a-his Cr 6
- Hv1a-lectin-his Cr 1
- Hv1a-lectin-his Cr 8
Cultivation
- Cultivation of ligated product.(7/16)
- Sample
- Hv1a-his Cr 5
- Hv1a-his Cr 7
- Hv1a-his Cr 10
- Hv1a-his Cr 11
- Hv1a-lectin-his Cr 2
- Hv1a-lectin-his Cr 3
- Hv1a-lectin-his Cr 7
July 18
PRC of IDT gblock fragment:
- Amplify the DNA to revise the mistake.
- Sample
- Hv1a-his-Sf1a NO.98351612
- Sf1a-his-OAIP NO.98351613
- OAIP-his-Hv1a NO.98351614
- Hv1a-lectin-his NO.98351615
- Sf1a-lectin-his NO.98351616
- OAIP-lectin-his NO.98351617
Miniprep
- Purify plasmid which have insert gene.(7/17)
- Sample
- Hv1a-his Cr 5
- Hv1a-his Cr 7
- Hv1a-his Cr 10
- Hv1a-his Cr 11
- Hv1a-lectin-his Cr 3
- Hv1a-lectin-his Cr 7
Cultivation
- Cultivation of ligated product.(7/17)
- Sample
- OAIP-lectin-his Cr 1
- OAIP-lectin-his Cr 2
Digestion
- Digest the plasmid (7/18) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Hv1a-his(EP)
- Hv1a-his(EP)
- Hv1a-his(EP)
- Hv1a-his(EP)
- Hv1a-lectin-his(EP)
- Hv1a-lectin-his(EP)
Gel Electrophoresis of PCR-Amplified gBlocks
- Sample
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- sf1a-lectin-his
- OAIP-lectin-his
Digestion
- Digest the gene fragment(7/18) and pSB1A3 with EcoRI and PstI to ligase the gene fragment into the pSB1A3.
- Sample
- Sf1a-his 1 7.18 (EP)
- Hv1a-lectin-his 4 7.18 (EP)
- OAIP-his 2 7.18 (EP)
- OAIP-lectin-his 5 7.18 (EP)
- Hv1a-his 3 7.18 (EP)
- Sf1a-lectin-his 6 7.18 (EP)
- pSB1C3 (EP)
Electrophoresis
- Electrophoresis of digested product to check the pSB1C3 digestion.
- Sample7/18 Digestion product
Ligation
- Ligase DNA fragment(7/18) into the pSB1C3
- Sample
- Sf1a-his 1 7.18 (EP)
- Hv1a-lectin-his 4 7.18 (EP)
- OAIP-his 2 7.18 (EP)
- OAIP-lectin-his 5 7.18 (EP)
- Hv1a-his 3 7.18 (EP)
- Sf1a-lectin-his 6 7.18 (EP)
- pSB1C3 (EP)
Electrophoresis
- check the ligation product.
- Sample7/18 Ligation product
July 19
Electrophoresis
- Electrophoresis of ligated product.
- Sample
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Transformation
- Transformation of ligated product to DH5α.
- Sample
- Hv1a-his 7/18 Ar
- Sf1a-his 7/18 Ar
- OAIP-his 7/8 Ar
- Hv1a-lectin-his 7/8 Ar
- sf1a-lectin-his 7/8 Ar
- OAIP-lectin-his 7/18 Ar
Miniprep
- Purify plasmid which have insert gene.(7/18)
- Sample
- OAIP-lectin-his Cr 7/19 1
- OAIP-lectin-his Cr 7/19 2
Digestion
- Digest the plasmid (7/19) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Hv1a-lectin-his Cr 1 (EP)
- Hv1a-lectin-his Cr 2 (EP)
Electrophoresis
- Electrophoresis of ligated product.
- Sample
- Hv1a-lectin-his Cr 7/19 1
- Hv1a-lectin-his Cr 7/19 2
Digestion
- Digest the plasmid (7/18) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Hv1a-his Cr 1
- Hv1a-his Cr 2
- Hv1a-his Cr 3
- Hv1a-his Cr 4
- Hv1a-lectin-his Cr 1
- Hv1a-lectin-his Cr 2
Electrophoresis
- Electrophoresis of digestion product.
- Sample
- Hv1a-his Cr 1
- Hv1a-his Cr 2
- Hv1a-his Cr 3
- Hv1a-his Cr 4
- Hv1a-lectin-his Cr 1
- Hv1a-lectin-his Cr 2
July 20
PCR
- PCR of ligated products(7/19) to amplify the insert gene.
- Sample
- Hv1a-his Ar 1~4
- Sf1a-his Ar 1~4
- OAIP-his Ar 1~4
- Hv1a-lectin-his Ar 1~4
- Sf1a-lectin-his Ar 1~4
- OAIP-lectin-his Ar 1~4
Digestion
- Digest the gene fragment(7/18) and pSB1A3 with EcoRI and PstI to ligase the gene fragment into the pSB1A3.
- Sample
- Hv1a-his 7/18 Cr(EP)
- Hv1a-his 7/18 Cr(EP)
- Hv1a-his 7/18 Cr(EP)
- Hv1a-his 7/18 Cr(EP)
- Hv1a-lectin-his 7/18 Cr(EP)
- Hv1a-lectin-his 7/18 Cr(EP)
- OAIP-lectin-his 7/18 Cr(EP)
Electrophoresis
- Electrophoresis of digestion product.
- Sample
- Hv1a-his 1~4 7/18(EP)
- Hv1a-lectin-his 1, 2 7/18 (EP)
- OAIP-lectin-his 7/19(EP)
- Hv1a-his 3 7/18
- Hv1a-lectin-his 2 7/18
Electrophoresis
- Check insert gene in E.coli.
- Sample
- Hv1a-his 1-1~4
- Hv1a-lectin-his 2-1~4
- Sf1a-his3-1~4
- Sf1a-lectin-his 4-1~4
- OAIP-his 5-1~4
- OAIP-lectin-his 6-1~4
Sequencing
- determine insert gene’s Sequence.
- Sample
- Hv1a-his 3(The result is correct)
- Hv1a-lectin-his 2
- OAIP-lectin-his 1
Ligation
- Ligase DNA fragment(7/18) into the pSB1A3
- Sample digestion product
- Hv1a-his 7/20 (EP)
- Hv1a-lectin-his 7/20 (EP)
- OAIP-lectin-his 7/20 (EP)
July 21
Cultivation
- Cultivation of ligated product.(7/20)
- Sample
- Hv1a-his Ar 2~4
- Sf1a-his Ar 1~4
- OAIP-his Ar 1.3.4
- Hv1a-lectin-his Ar 1~4
- Sf1a-lectin-his Ar 1~4
- OAIP-lectin-his Ar 1.3.4
Miniprep
- Purify plasmid which have insert gene.(7/21)
- Sample
- Hv1a-his Ar 2~4
- Sf1a-his Ar 1~4
- OAIP-his Ar 1.3.4
- Hv1a-lectin-his Ar 1~4
- Sf1a-lectin-his Ar 1~4
- OAIP-lectin-his Ar 1.3.4
Transformation
- Transformation of ligated product(7/20) to BL21-rosetta-gami
- Sample
- Hv1a-his Ar
- Hv1a-lectin-his Ar
- OAIP-lectin-his Ar
July 22
Digestion
- Digest the plasmid (7/21) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Hv1a-his Ar 2~4
- Sf1a-his Ar 1~4
- OAIP-his Ar 1.3.4
- Hv1a-lectin-his Ar 1~4
- Sf1a-lectin-his Ar 1~4
- OAIP-lectin-his Ar 1.3.4
Electrophoresis
- Electrophoresis of ligated product.
- Sample
- Hv1a-his Ar 2~4
- Sf1a-his Ar 1~4
- OAIP-his Ar 1.3.4
- Hv1a-lectin-his Ar 1~4
- Sf1a-lectin-his Ar 1~4
- OAIP-lectin-his Ar 1.3.4
Sequencing
- determine insert gene’s Sequence.
- Sample
- Hv1a-his (The result is correct)
- Hv1a -his (The result is correct)
- OAIP -his (The result is correct)
Digestion
- Digest the plasmid (7/21) with EcoRI and PstI to check whether the mini have insert gene fragment.
- Sample
- Sf1a-his Ar 1~4(EP)
- OAIP-his Ar 1.3.4(EP)
- OAIP-lectin-his Ar 1.3.4(EP)
Digestion
- Digest IDT gBlock fragment for ES because lectin have P Cut position.
- Sample
- Hv1a-lectin-his
- OAIP-lectin-his
- Sf1a-lectin-his
- Psb1A3
Electrophoresis
- Electrophoresis of ligated product.
- Sample digestion product
- Sf1a-his Ar 1~4(EP)
- OAIP-his Ar 1.3.4(EP)
- OAIP-lectin-his Ar 1.3.4(EP)
PRC of IDT gblock fragment
- Amplify the DNA to revise the mistake.
- Sample
- Hv1a-his
- Sf1a -his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Electrophoresis
- Electrophoresis of digested product to check insert gene.
- Sample
- Hv1a-lectin-his (ES)
- Sf1a-lectin-his (ES)
- OAIP-lectin-his (ES)
- [pSB1A3] (ES)
Ligation
- Ligase DNA fragment(7/18) into the pSB1A3
- Sample
- Hv1a-lectin-his (ES)
- Sf1a-lectin-his (ES)
- OAIP-lectin-his (ES)
- [pSB1A3] (ES)
Transformation
- Transformation of backbone plasmid into BL21-rosetta-gami check whether the competent cell is working.
- we choose pSB1A3 to transformation because BL21-rosetta-gami has Cr and Kr.
July 23
Electrophoresis
- After the PRC of IDT gBlock fragment, we electrophoresis samples to check the insert gene fragment.
- Sample
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
Digestion
- There is a fragment in the snow drop lectin identification with PstI. Therefore, if sequence have snow drop lectin, we digested the gene fragment(7/22) and pSB1A3 with EcoRI and Spe l to ligase the gene fragment into the pSB1A3. Others still digested with EcoRI and PstI.
- Sample
- Hv1a-his 3 7/18 (EP)
- Sf1a-his 1 7/8 (EP)
- OAIP-his 2 7/18 (EP)
- Hv1a-lectin-his 4 7/8 (ES)
- Sf1a-lectin-his 6 7/18 (ES)
- OAIP-lectin-his 5 7/18(ES)
- pSB1A3 (EP)
- pSB1A3 (ES)
Ligation
- Ligase DNA fragment(7/23) into the pSB1A3
- Sample
- Hv1a-his 3 7/18 (EP)
- Sf1a-his 1 7/8 (EP)
- OAIP-his 2 7/18 (EP)
- Hv1a-lectin-his 4 7/8 (ES)
- Sf1a-lectin-his 6 7/18 (ES)
- OAIP-lectin-his 5 7/18(ES)
- pSB1A3 (EP)
- pSB1A3 (ES)
Transformation
- Transformation of ligated product.
- Sample
- Hv1a-lectin-his Ar 7/22
- Sf1a-lectin-his Ar 7/22
- OAIP-lectin-his Ar 7/22
July 24
PCR
- PCR of ligated products(7/23) to amplify the insert gene.
- Sample
- Sf1a-lectin-his Ar 7/23 1~4
- OAIP-lectin-his Ar 7/23 5~8
- Hv1a-lectin-his Ar 7/23 9~12
Electrophoresis
- Electrophoresis of PCR products
- Sample
- Sf1a-lectin-his Ar 7/3 1~4
- OAIP-lectin-his Ar 7/23 5~8
- Hv1a-lectin-his Ar 7/23 9~12
Cultivation
- Cultivation of ligated product.(7/24)
- Sample
- Sf1a-lectin-his Ar 7/24 2.3
- OAIP-lectin-his Ar 7/24 5.7
- Hv1a-lectin-his Ar 7/24 9.12
July 25
Miniprep
- Purify plasmid which have insert gene.(7/24)
- Sample
- Sf1a-lectin-his Ar 7/24 2.3
- OAIP-lectin-his Ar 7/24 5.7
- Hv1a-lectin-his Ar 7/24 9.12
Digestion
- Digest the plasmid (7/25) with EcoRI and Spel to check whether the mini have insert gene fragment.
- Sample
- Sf1a-lectin-his Ar 2.3(ES)
- OAIP-lectin-his Ar 5.7(ES)
- Hv1a-lectin-his Ar 9.12(ES)
Electrophoresis
- Electrophoresis of ligated product.
- Sample
- Sf1a-lectin-his Ar 2.3(ES)
- OAIP-lectin-his Ar 5.7(ES)
- Hv1a-lectin-his Ar 9.12(ES)
Sequencing
- determine insert gene’s Sequence.
- Sample
- Hv1a-lectin-his (The result is correct)
- Hv1a-lectin-his (The result is correct)
- OAIP - lectin-his (The result is correct)
July 26
Prepare of CaCl2
- for competent cell
Rosetta-gami competent cell prepare
- for expression of disulfide bond.
Transformation
- Transformation of backbone plasmid into BL21-rosetta-gami check whether the competent cell is working.
- we choose pSB1A3 to transformation because BL21-rosetta-gami has Cr and Kr.
- Only write colonies. NO RFP EXPRESSION
July 27
Transformation
- Transformation of backbone plasmid into BL21-rosetta-gami check whether the competent cell is working.
- we choose pSB1A3 to transformation because BL21-rosetta-gami has Cr and Kr.
- Only write colonies. NO RFP EXPRESSION
Transformation
- Transformation of plasmid which have correct sequence to DH5α.
- Sample
- Sf1a -his
- OAIP-his
- Hv1a -his
July 28
Cultivation
- Cultivation of correct colonies. (7/27)
- Sf1a -his Ar
- OAIP-his Ar
- Hv1a -his Ar
Test competent cell
- BL21-rosetta-gami aren’t express RFP but write colonies. Therefore, we test competent cell whether they could save on Ar agar plate.
- After cultivation about 14 hours, there are many write colonies. We think them may be contamination during preparing competent cell.
July 29
Miniprep
- Purify plasmid which have correct insert gene(7/28) to conserve and transform to BL21-Rosetta-gami.
- Sf1a -his Ar
- OAIP-his Ar
- Hv1a -his Ar
Transformation
- Transformation of plasmid which have correct sequence to DH5α.
- Sample
- Sf1a-lectin-his 7/24 Ar
- OAIP-lectin-his 7/24 Ar
- Hv1a-lectin-his 7/24 Ar
July 30
Cultivation
- Cultivation of correct colonies. (7/29)
- Sf1a-lectin-his 7/29 Ar
- OAIP-lectin-his 7/29 Ar
- Hv1a-lectin-his 7/29 Ar
Miniprep
- Purify plasmid which have correct insert gene(7/30) to conserve and transform to BL21-Rosetta-gami.
- Sample
- Sf1a-lectin-his 7/29 Ar
- OAIP-lectin-his 7/29 Ar
- Hv1a-lectin-his 7/29 Ar
Preparation of Competent cell
- Rosetta-gami competent cell:
- Prepare the bacteria that can make disulfide bond
July 31
None
August 1
Transformation
- Transformation of pSB1A3 to test the competent cell work.
- sample
- PSB1A3
Transformation
- Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
- Sample
- Hv1a-his
- Sf1a-his
- OAIP-his
- Hv1a-lectin-his
- Sf1a-lectin-his
- OAIP-lectin-his
August 2
Transformation
- Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
- Sample
- Sf1a-his 7/28 Ar
- OAIP-his 7/28 Ar
- Hv1a-lectin-his 7/29 Ar
- Sf1a-lectin-his 7/29 Ar
August 3
Cultivation
- Cultivation of BL21-Rosetta-gam colonies for IPTG induction.
- sample
- Hv1a-his 8/1 Ar
- OAIP-lectin-his 8/1 Ar
Cultivation
- Cultivation of correct colonies again for mini extraction.
- Sample
- Sf1a-his 7/27 Ar
- OAIP-his 7/27 Ar
- Hv1a-lectin-his 7/24 Ar
- Sf1a-lectin-his 7/24 Ar
August 4
Miniprep
- Purify plasmid which have correct insert gene(8/3) to conserve and transform to BL21-Rosetta-gami.
- sample
- OAIP-his 8/3 Ar
- Sf1a-lectin-his 8/3 Ar
- Hv1a-lectin-his 8/3 Ar
Cultivation
- Cultivation of correct colonies again for mini extraction.
- Sample
- Sf1a-his 7/27 Ar
Transformation
- Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
- Sample
- OAIP-his 8/3 Ar
- Sf1a-lectin-his 8/3 Ar
- Hv1a-lectin-his 8/3 Ar
IPTG induction
- To find out the best condition for IPTG induction.
- Sample
- Hv1a-his 8/3 Ar
- OAIP-lectin-his 8/3 Ar
- Condition
0μM, 250μM, 500μM, 750μM, 1000μM - Add IPTG concentration and test OD600
0μM, 250μM, 500μM, 750μM, 1000μM
August 5
SDS-PAGE
- Check the protein production after IPTG condition.
- sample
- Marker
- OAIP-lectin-his 250μM 2hr
- OAIP-lectin-his 500μM 2hr
- OAIP-lectin-his 750μM 2hr
- OAIP-lectin-his 1000μM 2hr
- OAIP-lectin-his 0μM 4hr
- OAIP-lectin-his 250μM 4hr
- OAIP-lectin-his 500μM 4hr
- OAIP-lectin-his 750μM 4hr
- OAIP-lectin-his 1000μM 4hr
- Marker
- OAIP-lectin-his 250μM 6hr
- OAIP-lectin-his 500μM 6hr
- OAIP-lectin-his 750μM 6hr
- OAIP-lectin-his 1000μM 6hr
- OAIP-lectin-his 0μM 8hr
- OAIP-lectin-his 250μM 8hr
- OAIP-lectin-his 500μM 8hr
- OAIP-lectin-his 750μM 8hr
- OAIP-lectin-his 1000μM 8hr
August 6
None
August 7
None
August 8
None
August 9
None
August 10
None
August 11
None
August 12
None
August 13
None
August 14
Transformation
- Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
- Sample
- Sf1a-his 7/29 Ar
- OAIP-his 8/4 Ar
- Hv1a-his 7/29 Ar
- Sf1a-lectin-his 8/4 Ar
- OAIP-lectin-his 7/30 Ar
- Hv1a-lectin-his 8/4 Ar
PCR
- PCR of transformation colonies to check the base pair.
- Sample
- Hv1a-his Ar 1~6
- Hv1a-lectin-his Ar 1~6
- Sf1a-lectin-his Ar 1~6
- OAIP-lectin-his Ar 1~6
August 15
PCR
- PCR of ligated products to amplify the insert gene.
- Sample
- Hv1a-his 8/14 Ar 1~6
- Hv1a-lectin-his 8/14 Ar 1~6
- Sf1a- lectin-his 8/14 Ar 1~6
- Sf1a-lectin-his 8/4 Ar 1~6
- OAIP-lectin-his 7/30 Ar 1~6
- OAIP- lectin-his 8/14 Ar 1~6
Electrophoresis
- Electrophoresis of PCR products to check the insert gene base pair
- Sample
- Hv1a-his 8/15 Ar 1~6
- Hv1a-lectin-his 8/15 Ar 1~6
- Sf1a- lectin-his 8/15 Ar 1~6
- OAIP- lectin-his 8/15 Ar 1~6
Transformation
- Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
- Sample
- Sf1a-his 7/29 Ar
- OAIP-his 8/4 Ar
PCR
- PCR of ligated products to amplify the insert gene.
- Sample
- Sf1a- lectin-his 8/15 Ar 1~6
- OAIP- lectin-his 8/15 Ar 1~6
Electrophoresis
- Electrophoresis of PCR products to check the insert gene base pair.
- Sample
- Sf1a- lectin-his 8/15 Ar 1~6
- OAIP- lectin-his 8/15 Ar 1~6
SDS-PAGE
- Check the production of IPTG induction (8/4) again.
- No target protein expression.
August 16
Cultivation
- Cultivation of BL21-Rosetta-gami colonies for IPTG induction.
- Sample
- Hv1a-his 8/15 Ar
- Hv1a-lectin-his 8/15 Ar
- Sf1a- lectin-his 8/15 Ar
- OAIP-lectin-his 8/15 Ar
IPTG induction
- To check the protein expression for IPTG induction.
- Sample
- Hv1a-his 8/16 Ar
- Hv1a-lectin-his 8/16 Ar
- Sf1a- lectin-his 8/16 Ar
- OAIP-lectin-his 8/16 Ar
- Condition
0μM, 500μM - Add IPTG 500μM and test OD600 after 4hr.
Sonication and SDS-PAGE
- Sonication and run SDS-PAGE to check peptides expression level after the IPTG induction.
- Sample(8/16)
- Hv1a-his 0μM 2hr
- Hv1a-his 500μM 2hr
- Sf1a-lectin-his 0μM 4hr
- Sf1a-lectin-his 500μM 4hr
- Hv1a-lectin-his 0μM 2hr
- Hv1a-lectin-his 500μM 2hr
- OAIP-lectin-his 0μM 4hr
- OAIP-lectin-his 500μM 4hr
- Failed. Do it again
- Sample
- Hv1a-his 0μM 2hr
- Hv1a-his 500μM 2hr
- Sf1a-lectin-his 0μM 4hr
- Sf1a-lectin-his 500μM 4hr
- Hv1a-lectin-his 0μM 2hr
- Hv1a-lectin-his 500μM 2hr
- OAIP-lectin-his 0μM 4hr
- OAIP-lectin-his 500μM 4hr
August 17
IPTG induction
- To check the protein expression for IPTG induction.
- Sample
- Hv1a-his 8/17 Ar
- Hv1a-lectin-his
- Sf1a- lectin-his
- OAIP- lectin-his
- Condition
0μM, 500μM - Add IPTG 500μM and test OD600 after 4hr.
Sonication and SDS-PAGE
- Sonication and run SDS-PAGE to check peptides expression level after the IPTG induction.
- Sample
- Hv1a-his 0μM 2hr
- Hv1a-his 500μM 2hr
- Sf1a-lectin-his 0μM 4hr
- Sf1a-lectin-his 500μM 4hr
- Hv1a-lectin-his 0μM 2hr
- Hv1a-lectin-his 500μM 2hr
- OAIP-lectin-his 0μM 4hr
- OAIP-lectin-his 500μM 4hr
August 18
SDS-PAGE
- To Check protein disulfide bond folding.
- β-me make disulfide bond not folding.
- Sample
- Hv1a-lectin-his 0μM + β-me
- Hv1a-lectin-his 500μM + β-me
- Hv1a-lectin-his 500μM
- Sf1a-lectin-his 0μM + β-me
- Sf1a-lectin-his 500μM + β-me
- Sf1a-lectin-his 500μM
- OAIP-lectin-his 0μM + β-me
- OAIP-lectin-his 500μM + β-me
- OAIP-lectin-his 500μM
August 19
SDS-PAGE
- To Check protein disulfide bond folding with the induction of control, Rosetta-gami stain.
- β-me make disulfide bond not folding.
- Sample(8/17)
- Hv1a-lectin-his 0μM + β-me
- Hv1a-lectin-his 500μM + β-me
- Hv1a-lectin-his 500μM
- Sf1a-lectin-his 0μM + β-me
- Sf1a-lectin-his 500μM + β-me
- Sf1a-lectin-his 500μM
- Rosetta-gami + β-me
- OAIP-lectin-his 0μM + β-me
- OAIP-lectin-his 500μM + β-me
- OAIP-lectin-his 500μM
August 20
Sonication and SDS-PAGE
- Sonication and run SDS-PAGE to check peptides expression level after the IPTG induction.
- Sample
- Hv1a-his Ar
- Hv1a-his Ar
- Sf1a-lectin-his Ar
- Sf1a-lectin-his Ar
- Hv1a-lectin-his Ar
- Hv1a-lectin-his Ar
- OAIP-lectin-his Ar
- OAIP-lectin-his Ar
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-his Ar 8/20
- Hv1a-lectin-his Ar 8/20
- Sf1a-lectin-hiss Ar 8/20
- OAIP-lectin-his Ar 8/20
SDS-PAGE
- To Check protein disulfide bond folding with the induction of control, Rosetta-gami stain.
- Sample
- Hv1a-his 8/19
- Sf1a-his 8/19
- OAIP-his 8/19
August 21
IPTG Induction and Sonication
- To check the protein expression for IPTG induction. (Second round)
- Sample
- Hv1a-his Ar 8/20
- Hv1a-lectin-his Ar 8/20
- Sf1a-his Ar 8/17
- Sf1a-lectin-his Ar 8/20
- OAIP-his Ar 8/17
- OAIP-lectin-his Ar 8/20
- Condition
0μM, 500μM - Add IPTG 500μM and test OD600 after 4hr.
August 22
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- Hv1a-lectin-his 8/20 Ar
Cultivation
- Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
- Sample
- BL21-Rosetta-gami competent cell
- Cultivation 11 hours
IPTG Induction
- Prepare for the purify the protein from sample BL21-Rosetta-gami colonies.
- Sample
- Sf1a-his Ar 8/20 Ar
- Condition
0μM, 1000μM - Add IPTG 500μM and test OD600 after 4hr.
Sonication
- Sample
- Hv1a-lectin-his 8/22
- BL21-Rosetta-gami 8/22
- Sf1a-lectin-his 0μM 8/22
- Sf1a-lectin-his 1000μM 8/22
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
