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 C^r
• Hv1a-lectin-his 4 7.10 C^r
• OAIP-his 2 7.10 C^r
• OAIP-lectin-his 5 7.10 C^r
• Hv1a-his 3 7.10 C^r
• Sf1a-lectin-his 6 7.10 C^r

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 C^r
• Hv1a-lectin-his 4 7.10 C^r
• OAIP-his 2 7.10 C^r
• OAIP-lectin-his 5 7.10 C^r
• Hv1a-his 3 7.10 C^r
• Sf1a-lectin-his 6 7.10 C^r

July 13

PCR

• PCR of ligated products to amplify the insert gene.
• Sample
• Sf1a-his C^r 1~8
• Hv1a-lectin-his C^r 1~8
• OAIP-his C^r 1~8
• OAIP-lectin-his C^r 1~8
• Hv1a-his C^r 1~8
• Sf1a-lectin-his C^r 1~8

July 14

PCR

• PCR of ligated products to amplify the insert gene.
• Sample
• Sf1a-his C^r 1~4
• Hv1a-lectin-his C^r 1~4
• OAIP-his C^r 1~4
• OAIP-lectin-his C^r 1~4
• Hv1a-his C^r 1~4
• Sf1a-lectin-his C^r 1~4

Electrophoresis

• Electrophoresis of PCR products to check the insert gene base pair
• Sample
• Hv1a-his C^r 1~4
• Sf1a-his C^r 1~4
• OAIP-his C^r 1~4
• Hv1a-lectin-his C^r 1~4
• Sf1a-lectin-his C^r 1~4
• OAIP-lectin-his C^r 1~4

Cultivation

• Cultivation of ligated product.
• Sample
• Sf1a-his C^r 3
• Hv1a-lectin-his C^r 4
• OAIP-his C^r 1
• OAIP-lectin-his C^r 3
• Hv1a-his C^r 3
• Sf1a-lectin-his C^r 4

July 15

PCR

• PCR of ligated products to amplify the insert gene.
• Sample
• Sf1a-his C^r 1~8
• Hv1a-lectin-his C^r 1~8
• Hv1a-his C^r 1~6
• Sf1a-lectin-his C^r 1~3

Electrophoresis

• Electrophoresis of PCR products to check the insert gene base pair
• Sample
• Sf1a-his C^r 1~8
• Hv1a-lectin-his C^r 1~8
• Hv1a-his C^r 1~6
• Sf1a-lectin-his C^r 1~3

Transformation

• Transformation of Hv1a-lectin-his C^r, Hv1a-his C^r into BL21 again.

Miniprep

• Purify plasmid which have insert gene.(7/14)
• Sample
• Hv1a-his C^r
• Sf1a-his C^r
• OAIP-his C^r
• Hv1a-lectin-his C^r
• Sf1a-lectin-his C^r
• OAIP-lectin-his C^r

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 C^r 1~11
• Hv1a-lectin-his C^r 1~7

Electrophoresis

• Electrophoresis of PCR products to check the insert gene base pair.
• Sample
• Hv1a-his C^r 1~11
• Hv1a-lectin-his C^r 1~7

Cultivation

• Cultivation of ligated product.
• Sample
• Sf1a-his C^r 1
• Hv1a-his C^r 6
• Hv1a-lectin-his C^r 1
• Hv1a-lectin-his C^r 8
• Sf1a-lectin-his C^r 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 C^r 1
• Sf1a-lectin-his C^r 1
• Hv1a-his C^r 6
• Hv1a-lectin-his C^r 1
• Hv1a-lectin-his C^r 8

Cultivation

• Cultivation of ligated product.(7/16)
• Sample
• Hv1a-his C^r 5
• Hv1a-his C^r 7
• Hv1a-his C^r 10
• Hv1a-his C^r 11
• Hv1a-lectin-his C^r 2
• Hv1a-lectin-his C^r 3
• Hv1a-lectin-his C^r 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 C^r 5
• Hv1a-his C^r 7
• Hv1a-his C^r 10
• Hv1a-his C^r 11
• Hv1a-lectin-his C^r 3
• Hv1a-lectin-his C^r 7

Cultivation

• Cultivation of ligated product.(7/17)
• Sample
• OAIP-lectin-his C^r 1
• OAIP-lectin-his C^r 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 A^r
• Sf1a-his 7/18 A^r
• OAIP-his 7/8 A^r
• Hv1a-lectin-his 7/8 A^r
• sf1a-lectin-his 7/8 A^r
• OAIP-lectin-his 7/18 A^r

Miniprep

• Purify plasmid which have insert gene.(7/18)
• Sample
• OAIP-lectin-his C^r 7/19 1
• OAIP-lectin-his C^r 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 C^r 1 (EP)
• Hv1a-lectin-his C^r 2 (EP)

Electrophoresis

• Electrophoresis of ligated product.
• Sample
• Hv1a-lectin-his C^r 7/19 1
• Hv1a-lectin-his C^r 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 C^r 1
• Hv1a-his C^r 2
• Hv1a-his C^r 3
• Hv1a-his C^r 4
• Hv1a-lectin-his C^r 1
• Hv1a-lectin-his C^r 2

Electrophoresis

• Electrophoresis of digestion product.
• Sample
• Hv1a-his C^r 1
• Hv1a-his C^r 2
• Hv1a-his C^r 3
• Hv1a-his C^r 4
• Hv1a-lectin-his C^r 1
• Hv1a-lectin-his C^r 2

July 20

PCR

• PCR of ligated products(7/19) to amplify the insert gene.
• Sample
• Hv1a-his A^r 1~4
• Sf1a-his A^r 1~4
• OAIP-his A^r 1~4
• Hv1a-lectin-his A^r 1~4
• Sf1a-lectin-his A^r 1~4
• OAIP-lectin-his A^r 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 C^r(EP)
• Hv1a-his 7/18 C^r(EP)
• Hv1a-his 7/18 C^r(EP)
• Hv1a-his 7/18 C^r(EP)
• Hv1a-lectin-his 7/18 C^r(EP)
• Hv1a-lectin-his 7/18 C^r(EP)
• OAIP-lectin-his 7/18 C^r(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 A^r 2~4
• Sf1a-his A^r 1~4
• OAIP-his A^r 1.3.4
• Hv1a-lectin-his A^r 1~4
• Sf1a-lectin-his A^r 1~4
• OAIP-lectin-his A^r 1.3.4

Miniprep

• Purify plasmid which have insert gene.(7/21)
• Sample
• Hv1a-his A^r 2~4
• Sf1a-his A^r 1~4
• OAIP-his A^r 1.3.4
• Hv1a-lectin-his A^r 1~4
• Sf1a-lectin-his A^r 1~4
• OAIP-lectin-his A^r 1.3.4

Transformation

• Transformation of ligated product(7/20) to BL21-rosetta-gami
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• OAIP-lectin-his A^r

July 22

Digestion

• Digest the plasmid (7/21) with EcoRI and PstI to check whether the mini have insert gene fragment.
• Sample
• Hv1a-his A^r 2~4
• Sf1a-his A^r 1~4
• OAIP-his A^r 1.3.4
• Hv1a-lectin-his A^r 1~4
• Sf1a-lectin-his A^r 1~4
• OAIP-lectin-his A^r 1.3.4

Electrophoresis

• Electrophoresis of ligated product.
• Sample
• Hv1a-his A^r 2~4
• Sf1a-his A^r 1~4
• OAIP-his A^r 1.3.4
• Hv1a-lectin-his A^r 1~4
• Sf1a-lectin-his A^r 1~4
• OAIP-lectin-his A^r 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 A^r 1~4(EP)
• OAIP-his A^r 1.3.4(EP)
• OAIP-lectin-his A^r 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 A^r 1~4(EP)
• OAIP-his A^r 1.3.4(EP)
• OAIP-lectin-his A^r 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 C^r and K^r.

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 A^r 7/22
• Sf1a-lectin-his A^r 7/22
• OAIP-lectin-his A^r 7/22

July 24

PCR

• PCR of ligated products(7/23) to amplify the insert gene.
• Sample
• Sf1a-lectin-his A^r 7/23 1~4
• OAIP-lectin-his A^r 7/23 5~8
• Hv1a-lectin-his A^r 7/23 9~12

Electrophoresis

• Electrophoresis of PCR products
• Sample
• Sf1a-lectin-his A^r 7/3 1~4
• OAIP-lectin-his A^r 7/23 5~8
• Hv1a-lectin-his A^r 7/23 9~12

Cultivation

• Cultivation of ligated product.(7/24)
• Sample
• Sf1a-lectin-his A^r 7/24 2.3
• OAIP-lectin-his A^r 7/24 5.7
• Hv1a-lectin-his A^r 7/24 9.12

July 25

Miniprep

• Purify plasmid which have insert gene.(7/24)
• Sample
• Sf1a-lectin-his A^r 7/24 2.3
• OAIP-lectin-his A^r 7/24 5.7
• Hv1a-lectin-his A^r 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 A^r 2.3(ES)
• OAIP-lectin-his A^r 5.7(ES)
• Hv1a-lectin-his A^r 9.12(ES)

Electrophoresis

• Electrophoresis of ligated product.
• Sample
• Sf1a-lectin-his A^r 2.3(ES)
• OAIP-lectin-his A^r 5.7(ES)
• Hv1a-lectin-his A^r 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 C^r and K^r.
• 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 C^r and K^r.
• 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 A^r
• OAIP-his A^r
• Hv1a -his A^r

Test competent cell

• BL21-rosetta-gami aren’t express RFP but write colonies. Therefore, we test competent cell whether they could save on A^r 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 A^r
• OAIP-his A^r
• Hv1a -his A^r

Transformation

• Transformation of plasmid which have correct sequence to DH5α.
• Sample
• Sf1a-lectin-his 7/24 A^r
• OAIP-lectin-his 7/24 A^r
• Hv1a-lectin-his 7/24 A^r

July 30

Cultivation

• Cultivation of correct colonies. (7/29)
• Sf1a-lectin-his 7/29 A^r
• OAIP-lectin-his 7/29 A^r
• Hv1a-lectin-his 7/29 A^r

Miniprep

• Purify plasmid which have correct insert gene(7/30) to conserve and transform to BL21-Rosetta-gami.
• Sample
• Sf1a-lectin-his 7/29 A^r
• OAIP-lectin-his 7/29 A^r
• Hv1a-lectin-his 7/29 A^r

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 A^r
• OAIP-his 7/28 A^r
• Hv1a-lectin-his 7/29 A^r
• Sf1a-lectin-his 7/29 A^r

August 3

Cultivation

• Cultivation of BL21-Rosetta-gam colonies for IPTG induction.
• sample
• Hv1a-his 8/1 A^r
• OAIP-lectin-his 8/1 A^r

Cultivation

• Cultivation of correct colonies again for mini extraction.
• Sample
• Sf1a-his 7/27 A^r
• OAIP-his 7/27 A^r
• Hv1a-lectin-his 7/24 A^r
• Sf1a-lectin-his 7/24 A^r

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 A^r
• Sf1a-lectin-his 8/3 A^r
• Hv1a-lectin-his 8/3 A^r

Cultivation

• Cultivation of correct colonies again for mini extraction.
• Sample
• Sf1a-his 7/27 A^r

Transformation

• Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
• Sample
• OAIP-his 8/3 A^r
• Sf1a-lectin-his 8/3 A^r
• Hv1a-lectin-his 8/3 A^r

IPTG induction

• To find out the best condition for IPTG induction.
• Sample
• Hv1a-his 8/3 A^r
• OAIP-lectin-his 8/3 A^r
• 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 A^r
• OAIP-his 8/4 A^r
• Hv1a-his 7/29 A^r
• Sf1a-lectin-his 8/4 A^r
• OAIP-lectin-his 7/30 A^r
• Hv1a-lectin-his 8/4 A^r

PCR

• PCR of transformation colonies to check the base pair.
• Sample
• Hv1a-his A^r 1~6
• Hv1a-lectin-his A^r 1~6
• Sf1a-lectin-his A^r 1~6
• OAIP-lectin-his A^r 1~6

August 15

PCR

• PCR of ligated products to amplify the insert gene.
• Sample
• Hv1a-his 8/14 A^r 1~6
• Hv1a-lectin-his 8/14 A^r 1~6
• Sf1a- lectin-his 8/14 A^r 1~6
• Sf1a-lectin-his 8/4 A^r 1~6
• OAIP-lectin-his 7/30 A^r 1~6
• OAIP- lectin-his 8/14 A^r 1~6

Electrophoresis

• Electrophoresis of PCR products to check the insert gene base pair
• Sample
• Hv1a-his 8/15 A^r 1~6
• Hv1a-lectin-his 8/15 A^r 1~6
• Sf1a- lectin-his 8/15 A^r 1~6
• OAIP- lectin-his 8/15 A^r 1~6

Transformation

• Transformation of plasmid which have correct sequence to BL21-Rosetta-gami.
• Sample
• Sf1a-his 7/29 A^r
• OAIP-his 8/4 A^r

PCR

• PCR of ligated products to amplify the insert gene.
• Sample
• Sf1a- lectin-his 8/15 A^r 1~6
• OAIP- lectin-his 8/15 A^r 1~6

Electrophoresis

• Electrophoresis of PCR products to check the insert gene base pair.
• Sample
• Sf1a- lectin-his 8/15 A^r 1~6
• OAIP- lectin-his 8/15 A^r 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 A^r
• Hv1a-lectin-his 8/15 A^r
• Sf1a- lectin-his 8/15 A^r
• OAIP-lectin-his 8/15 A^r

IPTG induction

• To check the protein expression for IPTG induction.
• Sample
• Hv1a-his 8/16 A^r
• Hv1a-lectin-his 8/16 A^r
• Sf1a- lectin-his 8/16 A^r
• OAIP-lectin-his 8/16 A^r
• 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 A^r
• 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 A^r
• Hv1a-his A^r
• Sf1a-lectin-his A^r
• Sf1a-lectin-his A^r
• Hv1a-lectin-his A^r
• Hv1a-lectin-his A^r
• OAIP-lectin-his A^r
• OAIP-lectin-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r 8/20
• Hv1a-lectin-his A^r 8/20
• Sf1a-lectin-hiss A^r 8/20
• OAIP-lectin-his A^r 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 A^r 8/20
• Hv1a-lectin-his A^r 8/20
• Sf1a-his A^r 8/17
• Sf1a-lectin-his A^r 8/20
• OAIP-his A^r 8/17
• OAIP-lectin-his A^r 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 A^r

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 A^r
• 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 A^r 8/20
• Sf1a-lectin-his A^r 8/20
• Add 1000μM IPTG and test OD₆₀₀

Sonication

• Sample
• Hv1a-his A^r 8/23
• Sf1a-lectin-his A^r 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 A^r 8/20
• Sf1a-lectin-his A^r 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 A^r 8/24
• Sf1a-lectin-his A^r 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 A^r 8/15
• Hv1a-lectin-his A^r 8/15
• Sf1a-lectin-his A^r 8/15
• OAIP-lectin-his A^r 8/15

Sonication

• Sample
• Sf1a-his A^r 8/24
• Sf1a-lectin-his A^r 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 A^r 8/17
• OAIP-his A^r 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 A^r 8/26
• Hv1a-lectin-his A^r 8/26
• Sf1a-lectin-his A^r 8/26
• OAIP-lectin-his A^r 8/26
• Add 500μM IPTG
• Take the following samples to go on the experience first. The others storage in 4℃ refrigerator.
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26

SDS-PAGE

• Check the protein in pellet or in solution.
• Sample
• Hv1a-his A^r 8/26(with urea)
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26(with urea)
• Hv1a-lectin-his A^r 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 A^r 8/26
• Hv1a-lectin-his A^r 8/26
• Sf1a-lectin-his A^r 8/26
• OAIP-lectin-his A^r 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 A^r 8/26(with urea)
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26(with urea)
• Hv1a-lectin-his A^r 8/26
• Sf1a-lectin-his A^r 8/26(with urea)
• Sf1a-lectin-his A^r 8/26
• OAIPlectin-his A^r 8/26(with urea)
• OAIP-lectin-his A^r 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 A^r
• Hv1a-lectin-his A^r

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 A^r 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 A^r 8/17

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r 8/15

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-lectin-his A^r 8/15
• Sf1a-lectin-his A^r 8/15
• OAIP-lectin-his A^r 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 A^r 8/29
• Sf1a-lectin-his A^r 8/29
• OAIP-lectin-his A^r 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 A^r
• Hv1a-lectin-his A^r
• OAIP-his A^r
• Sf1a-his A^r

IPTG Induction

• Prepare for the purify the protein from sample BL21-Rosetta-gami colonies.
• Sample
• Hv1a-his A^r
• OAIP-his A^r
• Sf1a-his A^r
• Add IPTG 500μM and test OD₆₀₀ after 4hr.

Sonication

• Sample
• Hv1a-his A^r
• OAIP-his A^r
• Sf1a-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Sonication

• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

September 2

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• 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 A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

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 A^r
• OAIP-lectin-his A^r
• 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 A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Concentrate protein

• Increase the concentration of protein
• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

September 4

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• 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 A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• Add IPTG 500μM and test OD₆₀₀ after 4hr.

Sonication

• Sample
• Hv1a-his
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

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 A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Concentrate protein

• Increase the concentration of protein
• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Protein quantification Braford method

• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

September 6

Protein purification

• The buffer contains the urea
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• 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 A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 7

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• 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 A^r
• Sf1a-lectin-his A^r

UV test

• Test degradation rate
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Sonication

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 8

Protein purification

• The buffer contains the urea
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• 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 A^r
• Hv1a-lectin-his A^r

Cultivation and IPTG induction

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r
• 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 A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Sonication

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r
• 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 A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 9

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r
• 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 A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

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 A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

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 A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

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 A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 26

None

September 27

None

September 28

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• OAIP-his A^r

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 A^r

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