Team:NCTU Formosa/Notebook

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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