《结构免疫学》课程教学资源（实验室SOP）6 附录

实验人员注意以下事项：1由于衍射仪测角头电机比较小，如果手动上样的话很容易把位置碰歪，所以目前只能机械手上样，建议各位老师将晶体样品保存在puck里，如果没有puck，可以提前到线站借用我们线站的puck将晶体样品转移到puck里再通过机械手上样。2.如果在实验的过程中发生故障，一定要第一时间给值班人员打电适，不能自行去处理。3.建议在EIGER16M探测器上收数据的策略是：0.1~0.3度一张，曝光时间0.05秒一张。这样收一套360度的完整数据（3600张衍射图）大概只需要3分钟4.一套3600张衍射图的数据大小是25GB左右，建议带一个存储空间大的硬盘拷贝数据，可以边实验边拷贝数据。减少拷贝数据等待时间。5.数据处理建议使用XDS。6.目前线站的新系统还需要进一步优化调试，能量暂时固定在12.660KeV。6.2.6实验室载体CloningSequencingSequencingNameBacknoneDescriptionFusion TagResistanceFramePrimer 1Primer2Ndel-T7T7Sall,Notl-Sall-V1pET30aKannoneNotlXhol-His6-PromoterTerminatorstopNdel-His8-Sall-T7T7V2pET30a Sall,Notl-KannoneNotlPromoterTerminatorstopNdel-His8-Sall-T7T7GB1-TEV-His8-GB1-V3KanpET30aNotlTEVSall,Notl-PromoterTerminatorstopNdel-His8-GB1-TEV-T7T7Sall-His8-GB1-V4pET30aSall,Notl-KanNotlTEVPromoterTerminatorXhol-His6-stopNdel-His8-TEV-Sall-T7T7MGSS-His8-V5pET30aKanNotlTEVSall,Notl-PromoterTerminatorstopNdel-His8-TEV-Sall-T7T7MGSS-His8-V6pET30aSall,Notl-KanNotlTEVPromoterTerminatorXhol-His6-stop Ndel-His8-SUMO-T7T7Sall-MGSS-His8-V7TEV-pET30aKanNotlSUMO-TEVPromoterTerminatorSall,Notl-Xhol-stopNdel-His8-VHb-TEV-Sall-His8-VHb-T7T7V8pET30aKanSall,Notl-NotlTEVPromoterTerminatorstopNdel-His8-VHb-TEV-T7Sall-His8-VHb-T7V9Sall,Notl-KanpET30aNotlTEVPromoterTerminatorXhol-His6-stopV10Sall-His8-GST-TEVT7T7pET30aNdel-His8-KanGST-TEV-NotlPromoterTerminator

实验人员注意以下事项： 1. 由于衍射仪测角头电机比较小，如果手动上样的话很容易把位置碰歪，所以目前只能机械手上样，建议各位老师将 晶体样品保存在puck里，如果没有puck，可以提前到线站借用我们线站的puck将晶体样品转移到puck里再通过机械手上 样。 2. 如果在实验的过程中发生故障，一定要第一时间给值班人员打电话，不能自行去处理。 3. 建议在EIGER 16 M探测器上收数据的策略是：0.1~0.3度一张，曝光时间0.05秒一张。这样收一套360度的完整数据 （3600张衍射图）大概只需要3分钟。 4. 一套3600张衍射图的数据大小是25 GB左右，建议带一个存储空间大的硬盘拷贝数据，可以边实验边拷贝数据。减 少拷贝数据等待时间。 5. 数据处理建议使用XDS。 6. 目前线站的新系统还需要进一步优化调试，能量暂时固定在12.660 Kev。 6.2.6 实验室载体 Name Backnone Description Cloning Frame Fusion Tag Sequencing Primer 1 Sequencing Primer2 Resistance V1 pET30a NdeI￾SalI,NotI￾XhoI-His6- stop SalI￾NotI none T7 Promoter T7 Terminator Kan V2 pET30a NdeI-His8- SalI,NotI￾stop SalI￾NotI none T7 Promoter T7 Terminator Kan V3 pET30a NdeI-His8- GB1-TEV￾SalI,NotI￾stop SalI￾NotI His8-GB1- TEV T7 Promoter T7 Terminator Kan V4 pET30a NdeI-His8- GB1-TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI His8-GB1- TEV T7 Promoter T7 Terminator Kan V5 pET30a NdeI-His8- TEV￾SalI,NotI￾stop SalI￾NotI MGSS-His8- TEV T7 Promoter T7 Terminator Kan V6 pET30a NdeI-His8- TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI MGSS-His8- TEV T7 Promoter T7 Terminator Kan V7 pET30a NdeI-His8- SUMO￾TEV￾SalI,NotI￾XhoI-stop SalI￾NotI MGSS-His8- SUMO-TEV T7 Promoter T7 Terminator Kan V8 pET30a NdeI-His8- VHb-TEV￾SalI,NotI￾stop SalI￾NotI His8-VHb￾TEV T7 Promoter T7 Terminator Kan V9 pET30a NdeI-His8- VHb-TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI His8-VHb￾TEV T7 Promoter T7 Terminator Kan V10 pET30a NdeI-His8- GST-TEV￾SalI￾NotI His8-GST-TEV T7 Promoter T7 Terminator Kan

Sall,Notl-stopNdel-His8-GST-TEV-T7T7Sall-V11pET30aSall,Notl-His8-GST-TEVKanNotlPromoterTerminatorXhol-His6-stopNdel-His7-T7T7TRX-TEV-Sall-His7-TRX-V12KanpET30aNotlTEVSall,Notl-PromoterTerminatorstopNdel-His7-TRX-TEV-T7T7Sall-His7-TRX-V13pET30aSall,Notl-KanNotlTEVPromoterTerminatorXhol-His6-stopNdel-His8-T7T7MBP-Sall-V14His8-MBPpET30aKanNotlSall,Notl-PromoterTerminatorstopNdel-His8-T7T7tag2-TEV-Sall-V15pET30aHis8-tag2-TEVKanNotlSall,Notl-PromoterTerminatorstopNdel-His8-tag2-TEV-T7T7Sall-V16His8-tag2-TEVKanpET30aSall,Notl-NotlPromoterTerminatorXhol-His6-stopNdel-DsbA-T7TEV-Sall-T7V17DsbA-TEVpET30aKanSall,Notl-NotlPromoterTerminatorXhol-His6-stopNdel-MBP-T7T7Sall-MBPV18Sall,Notl-KanpET30aNotlPromoterTerminatorXhol-His6-stopNdel-His8-NusA-T7T7Sall-His8-NusA-V19TEV-KanpET30aNotlTEVPromoterTerminatorSall,Notl-stopNdel-His8-GB1-TEV-Sall,Notl-T7T7Sall-His8-GB1-AcidicV20KanpET30aNotlTEVPeptide-PromoterTerminatorLacZ-Xhol-His6-stopNdel-His8-MBP-T7T7Sall-His8-MBP-helicalV21KanpET30aNotllinker-helical linkerPromoterTerminatorSall,Notl-stop

SalI,NotI￾stop V1 1 pET30a NdeI-His8- GST-TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI His8-GST-TEV T7 Promoter T7 Terminator Kan V12 pET30a NdeI-His7- TRX-TEV￾SalI,NotI￾stop SalI￾NotI His7-TRX￾TEV T7 Promoter T7 Terminator Kan V13 pET30a NdeI-His7- TRX-TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI His7-TRX￾TEV T7 Promoter T7 Terminator Kan V14 pET30a NdeI-His8- MBP￾SalI,NotI￾stop SalI￾NotI His8-MBP T7 Promoter T7 Terminator Kan V15 pET30a NdeI-His8- tag2-TEV￾SalI,NotI￾stop SalI￾NotI His8-tag2-TEV T7 Promoter T7 Terminator Kan V16 pET30a NdeI-His8- tag2-TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI His8-tag2-TEV T7 Promoter T7 Terminator Kan V17 pET30a NdeI￾DsbA￾TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI DsbA-TEV T7 Promoter T7 Terminator Kan V18 pET30a NdeI￾MBP￾SalI,NotI￾XhoI-His6- stop SalI￾NotI MBP T7 Promoter T7 Terminator Kan V19 pET30a NdeI-His8- NusA￾TEV￾SalI,NotI￾stop SalI￾NotI His8-NusA￾TEV T7 Promoter T7 Terminator Kan V20 pET30a NdeI-His8- GB1-TEV￾SalI,NotI￾Acidic Peptide￾LacZ￾XhoI-His6- stop SalI￾NotI His8-GB1- TEV T7 Promoter T7 Terminator Kan V21 pET30a NdeI-His8- MBP￾helical linker - SalI,NotI￾stop SalI￾NotI His8-MBP￾helical linker T7 Promoter T7 Terminator Kan

Ndel-MBP-Sall-M13R-MBPV22PMALSall,Notl-M13F-pUCAmpNotlpUCXhol-LacZ-stopNdel-MBP-Sall-M13R-MBPV23pMALSall,Notl-M13F-pUCAmpNotlpUCXhol-LacZ-stopNdel-MBP-Sall-M13R-V24MBPpMALSall,Notl-M13F-pUCAmpNotlpUCXhol-LacZ-stopNdel-MBP-Sall-M13R-V25pMALSall,Notl-MBPM13F-pUCAmpNotlpUCXhol-LacZ-stopNdel-MBP-Sall-M13R-MBPV26pMALSall,Notl-M13F-pUCAmpNotlpUCXhol-LacZ-stopNdel-MBP-Sall-M13R-V27pMALSall,Notl-MBPM13F-pUCAmpNotlpUCXhol-LacZ-stopNdel-MBP-Sall-T7T7V28pET30aSall,Notl-MBPKanNotlPromoterTerminatorXhol-His6-stopNdel-T7T7MBP-TEV-Sall-V29pET30aMBP-TEVKanNotlPromoterSall,Notl-TerminatorstopNdel-His8-Sall-T7T7MBP-TEV-His8-MBP-V29HpET30aKanNotlSall,Notl-TEVPromoterTerminatorstopNdel-MBP-TEV-T7T7Sall-V30pET30aSall,Notl-MBP-TEVKanNotlPromoterTerminatorXhol-His6-stopNcol-SP-Sall-V31P14A26pLEXSY-BleSall,Notl-Signal PeptideAmp/Ble/TetNotlHis6-stopNcol-SP-Strep Tag-Sall-SP-Strep Tag-V32P14A26pLEXSY-BleHis8-TEV-Amp/Ble/TetNotlHis8-TEVSall,Notl-His6-stopNcol-Sall-V33P14A26pLEXSY-BleSall,Notl-Amp/Ble/TetnoneNotlHis8-stop

V22 pMAL NdeI￾MBP￾SalI,NotI￾XhoI￾LacZ-stop SalI￾NotI MBP M13R￾pUC M13F-pUC Amp V23 pMAL NdeI￾MBP￾SalI,NotI￾XhoI￾LacZ-stop SalI￾NotI MBP M13R￾pUC M13F-pUC Amp V24 pMAL NdeI￾MBP￾SalI,NotI￾XhoI￾LacZ-stop SalI￾NotI MBP M13R￾pUC M13F-pUC Amp V25 pMAL NdeI￾MBP￾SalI,NotI￾XhoI￾LacZ-stop SalI￾NotI MBP M13R￾pUC M13F-pUC Amp V26 pMAL NdeI￾MBP￾SalI,NotI￾XhoI￾LacZ-stop SalI￾NotI MBP M13R￾pUC M13F-pUC Amp V27 pMAL NdeI￾MBP￾SalI,NotI￾XhoI￾LacZ-stop SalI￾NotI MBP M13R￾pUC M13F-pUC Amp V28 pET30a NdeI￾MBP￾SalI,NotI￾XhoI-His6- stop SalI￾NotI MBP T7 Promoter T7 Terminator Kan V29 pET30a NdeI￾MBP-TEV￾SalI,NotI￾stop SalI￾NotI MBP-TEV T7 Promoter T7 Terminator Kan V29H pET30a NdeI-His8- MBP-TEV￾SalI,NotI￾stop SalI￾NotI His8-MBP￾TEV T7 Promoter T7 Terminator Kan V30 pET30a NdeI￾MBP-TEV￾SalI,NotI￾XhoI-His6- stop SalI￾NotI MBP-TEV T7 Promoter T7 Terminator Kan V31 pLEXSY-Ble NcoI-SP￾SalI,NotI￾His6-stop SalI￾NotI Signal Peptide P14 A26 Amp/Ble/Tet V32 pLEXSY-Ble NcoI-SP￾Strep Tag￾His8-TEV￾SalI,NotI￾His6-stop SalI￾NotI SP-Strep Tag￾His8-TEV P14 A26 Amp/Ble/Tet V33 pLEXSY-Ble NcoI￾SalI,NotI￾His8-stop SalI￾NotI none P14 A26 Amp/Ble/Tet

Ncol-StrepTag-Sall-P14V34pLEXSY-BleA26Strep TagAmp/Ble/TetNotlSall,Notl-His8-stopNcol-His8-GST-TEV-Sall-P14V35pLEXSY-BleHis8-GST-TEVA26Amp/Ble/TetNotlSall,Notl-stopNcol-SP-Strep-His8-Sall-SP-Strep-His8-P14V36pLEXSY-BleTEV-A26Amp/Ble/TetNotlTEVSall,Notl-His6-stopNcol-Strep-His8-Sall-Strep-His8-P14V37TEV-A26Amp/Ble/TetpLEXSY-BleNotlTEVSall,Notl-His6-stopNcol-His8-T7T7GB1-TEV-Sall-His8-GB1-V40pTT5KanTEVSall,Notl-NotlPromoterTerminatorstopNcol-His6-Strep-His8-Sall-His6-Strep-V41P14pTT5TEV-A26Amp/Ble/TetNotlHis8-TEVSall,Notl-stopNcol-Strep-His8-Sall-Strep-His8-P14V42PTTSTEV-A26Amp/Ble/TetNotlTEVSall,Notl-stopEcoRV-SP-Strep-His8-Sall-SP-Strep-His8-V51pFabTEV-unknownunknownAmpNotlTEVSall,Notl-His6-stopEcoRV-SP-His6-TEV-Sall-V52pFabSP-His6-TEVunknownunknownAmpSall,Notl-NotlstopEcoRV-SP-Sall-His6-TEV-V53pFabSP-His6-TEVunknownunknownAmpNotlSall,Notl-stopEcoRV-SP-Sall-His6-TEV-V54pFabSP-His6-TEVunknownunknownAmpNotlSall,Notl-Strep-stopNdel-Sall-M13R-MBP-TEV-V55PMALMBP-TEVM13F-pUCAmpSall,Notl-NotlpUCstopLexA-Sall-ForV56pMW103Sall,Notl-LexAunknownKanunknownNotlY2HstopV57Ndel-Sall-T7T7pET30ahDim2KanhDim2-NotlPromoterTerminatorSall,Notl-

V34 pLEXSY-Ble NcoI-Strep Tag￾SalI,NotI￾His8-stop SalI￾NotI Strep Tag P14 A26 Amp/Ble/Tet V35 pLEXSY-Ble NcoI-His8- GST-TEV￾SalI,NotI￾stop SalI￾NotI His8-GST-TEV P14 A26 Amp/Ble/Tet V36 pLEXSY-Ble NcoI-SP￾Strep-His8- TEV￾SalI,NotI￾His6-stop SalI￾NotI SP-Strep-His8- TEV P14 A26 Amp/Ble/Tet V37 pLEXSY-Ble NcoI￾Strep-His8- TEV￾SalI,NotI￾His6-stop SalI￾NotI Strep-His8- TEV P14 A26 Amp/Ble/Tet V40 pTT5 NcoI-His8- GB1-TEV￾SalI,NotI￾stop SalI￾NotI His8-GB1- TEV T7 Promoter T7 Terminator Kan V41 pTT5 NcoI-His6- Strep-His8- TEV￾SalI,NotI￾stop SalI￾NotI His6-Strep￾His8-TEV P14 A26 Amp/Ble/Tet V42 pTT5 NcoI￾Strep-His8- TEV￾SalI,NotI￾stop SalI￾NotI Strep-His8- TEV P14 A26 Amp/Ble/Tet V51 pFab EcoRV-SP￾Strep-His8- TEV￾SalI,NotI￾His6-stop SalI￾NotI SP-Strep-His8- TEV unknown unknown Amp V52 pFab EcoRV-SP￾His6-TEV￾SalI,NotI￾stop SalI￾NotI SP-His6-TEV unknown unknown Amp V53 pFab EcoRV-SP￾His6-TEV￾SalI,NotI￾stop SalI￾NotI SP-His6-TEV unknown unknown Amp V54 pFab EcoRV-SP￾His6-TEV￾SalI,NotI￾Strep-stop SalI￾NotI SP-His6-TEV unknown unknown Amp V55 pMAL NdeI￾MBP-TEV￾SalI,NotI￾stop SalI￾NotI MBP-TEV M13R￾pUC M13F-pUC Amp V56 pMW103 LexA￾SalI,NotI￾stop SalI￾NotI LexA unknown unknown Kan For Y2H V57 pET30a NdeI￾hDim2- SalI,NotI￾SalI￾NotI hDim2 T7 Promoter T7 Terminator Kan

Xhol-His6-stopNdel-His8-TEV-T7T7Sall-V59Sall,Notl-His8-TEVpET30aKanNotlPromoterTerminatorLysozyme-stopNdel-Lysozyme-T7T7Sall-V60Sall,Notl-pET30aLysozymeKanNotlPromoterTerminatorXhol-His6-stopHis6-TEV-Sall-V61pEU-01His6-TEVSall,Notl-unknownunknownAmpNotlstopNdel-MBP-T7T7Sall-Sall,Notl-V62MBPpET30aKanNotllysozyme-PromoterTerminatorXhol-His6-stopNdel-MBP-TEV-T7T7Sall,Notl-Sall-V63MBP-TEVpET30aKanNotlLysozyme-PromoterTerminatorXhol-His6-stopNdel-His8-MBP-TEV-T7T7Sall-His8-MBP-V64KanpET30aSall,Notl-NotlTEVPromoterTerminatorLysozyme-stopGal1-B42-HASall-ForGal1-B42-HAepitope-V65pJG4-5unknownunknownAmpEcoRI-NotlY2HepitopeSall,Notl-stopNdel-T4LS5-T7T7Sall-V66Sall,Notl-T4LS5KanpET30aNotlPromoterTerminatorXhol-His6-stopNdel-His8-Ncol-TEV-Sall-T7T7V67PEU-01His8-TEVKanNotlSall,Notl-PromoterTerminatorstopNdel-T7T7Sall,Notl-Sall-V71KanpET30anoneNotlPromoterXhol-His6-TerminatorstopNdel-Sall-T7T7V72pET30aSall,Notl-KannoneNotlPromoterTerminatorStrep-stopNdel-Sall,Notl-Sall-V77pBADpBADforpBADrevnoneAmpNotlXhol-His6-stopV78pBAD-V28ENdel-Sall-MBPpBAD forpBADrevAmpMBP-Notl

XhoI-His6- stop V59 pET30a NdeI-His8- TEV￾SalI,NotI- Lysozyme￾stop SalI￾NotI His8-TEV T7 Promoter T7 Terminator Kan V60 pET30a NdeI￾Lysozyme￾SalI,NotI￾XhoI-His6- stop SalI￾NotI Lysozyme T7 Promoter T7 Terminator Kan V61 pEU-01 His6-TEV￾SalI,NotI￾stop SalI￾NotI His6-TEV unknown unknown Amp V62 pET30a NdeI￾MBP￾SalI,NotI￾lysozyme￾XhoI-His6- stop SalI￾NotI MBP T7 Promoter T7 Terminator Kan V63 pET30a NdeI￾MBP-TEV￾SalI,NotI- Lysozyme￾XhoI-His6- stop SalI￾NotI MBP-TEV T7 Promoter T7 Terminator Kan V64 pET30a NdeI-His8- MBP-TEV￾SalI,NotI- Lysozyme￾stop SalI￾NotI His8-MBP￾TEV T7 Promoter T7 Terminator Kan V65 pJG4-5 Gal1-B42- HA epitope￾EcoRI￾SalI,NotI￾stop SalI￾NotI Gal1-B42-HA epitope unknown unknown Amp For Y2H V66 pET30a NdeI￾T4LS5- SalI,NotI￾XhoI-His6- stop SalI￾NotI T4LS5 T7 Promoter T7 Terminator Kan V67 pEU-01 NdeI-His8- NcoI-TEV￾SalI,NotI￾stop SalI￾NotI His8-TEV T7 Promoter T7 Terminator Kan V71 pET30a NdeI￾SalI,NotI￾XhoI-His6- stop SalI￾NotI none T7 Promoter T7 Terminator Kan V72 pET30a NdeI￾SalI,NotI￾Strep-stop SalI￾NotI none T7 Promoter T7 Terminator Kan V77 pBAD NdeI￾SalI,NotI￾XhoI-His6- stop SalI￾NotI none pBAD for pBAD rev Amp V78 pBAD-V28E NdeI￾MBP￾SalI￾NotI MBP pBAD for pBAD rev Amp