This protocol is designed for the isolation of 96 plasmid DNA samples from 1.3ml bacterial cultures. Yield is up to 6µg.

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1 ====================================================== Date : Monday, November 14, :31 File Name :.PRO Full Name : Abstract : QIAprepTurbo 96 QIAsoft 4.1.3, Version 6.1, , EW, PK, HL, CK, KW This protocol is designed for the isolation of 96 plasmid DNA samples from 1.3ml bacterial cultures. Yield is up to 6µg. The protocol is written for BR8000R with 0.9mm Steel Probes and 2500µl Syringes. It s certified for version V0. It is ready to run on version V4. The protocol is not designed for versions V1, V2, V3, V5 and V6. Protocol Adjustments: e.g. date, initials, adjustment ====================================================== Variables defined for this protocol: (Comment, Name, Value, Default Value, Type, Min. Range, Max. Range) (SYS) Textual Indicator, BY_NAME, BY_NAME, BY_NAME, text (SYS) Textual Indicator, BY_NUMBER, BY_NUMBER, BY_NUMBER, text (SYS) Current Dest. Position, CurDPos,,, text (SYS) Current Source Position, CurSPos,,, text (SYS) Data Directory, DataDir, C:\Program Files\QIAsoft 4.1\Data\, C:\Program Files\QIAsoft 4.1\Data\, text (User) For Loop Counter, DryCounter, 5, 0, num, 0, 0 (SYS) Dilutor1 Size, DS1, ul, ul, num, 0.00, 0.00 (SYS) Dilutor2 Size, DS2, ul, ul, num, 0.00, 0.00 (SYS) Dilutor3 Size, DS3, ul, ul, num, 0.00, 0.00 (SYS) Dilutor4 Size, DS4, ul, ul, num, 0.00, 0.00 (SYS) Dilutor5 Size, DS5, ul, ul, num, 0.00, 0.00 (SYS) Dilutor6 Size, DS6, ul, ul, num, 0.00, 0.00 (SYS) Dilutor7 Size, DS7, ul, ul, num, 0.00, 0.00 (SYS) Dilutor8 Size, DS8, ul, ul, num, 0.00, 0.00 Elution Volume, ElutionVolume, 140µl, 125µl, num, 50, 150 (SYS) EQUAL-Operator ==, EQUAL, 4, 4, num, 4, 4 (SYS) Boolean Constant, FALSE, 0, 0, num, 0, 0 (SYS) GREATER or EQUAL-Operator >=, GREATEQ, 3, 3, num, 3, 3 (SYS) GREATER-Operator >, GREATER, 2, 2, num, 2, 2 (SYS) LOWER or EQUAL-Operator <=, LOWEQ, 1, 1, num, 1, 1 (SYS) LOWER-Operator <, LOWER, 0, 0, num, 0, 0 (SYS) Main Directory, MainDir, C:\Program Files\QIAsoft 4.1\, C:\Program Files\QIAsoft 4.1\, text (SYS) Number Of Samples, NoSam, 96, 96, num, 0, 0 (SYS) NOT-EQUAL-Operator <>, NOTEQUAL, 5, 5, num, 5, 5 (SYS) Position Counter, PosCount, 0, 0, num, 0, 0 Report File, ReportFile,,, text (SYS) Command Return Value, Return, 0, Return, text (SYS) Sample Positions, Samples,,, text State of System Liquid, SLstate, 0, 0, num, 0, 0 (SYS) Seconds until timer has elapsed, TimerValue, , 0, num, 0, 0 Total Elution Volume, TotalElutionVolume, 16.1ml, 10.0ml, num, 0.0, (SYS) Boolean Constant, TRUE, 1, 1, num, 1, 1 (SYS) Userdata Directory, UserdataDir, C:\Program Files\QIAsoft 4.1\UserData\, C:\Program Files\QIAsoft 4.1\UserData\, text State of Vacuum Trap Sensor, vacstate, 0, 0, num, 0, 0 State of Waste Bottle, wastestate, 1, 1, num, 0, 0 (SYS) Table Information, Info, tab Setting Volume Table, SettingVolumeTable, tab Protocol commands: ( 1) REMARK: QIAsoft 4.1.3, Version 6.1, , EW, PK, HL, CK, KW Page 1

2 ( 2) REMARK: BR8000R, 0.9mm Steel Probes, 2500µl Syringes ( 3) REMARK: ( 4) ROBOT CONTROL: Call method 'InitRobot' on device 'Robot' ( 5) DO LOOP ( 6) USER INTERACTION: Display Message: General Interactions Refill System Liquid Bottle. ( 7) DIGITAL IO: Read input from device 'System Liquid Sensor' into variable 'SLstate' ( 8) WHILE SLstate IS EQUAL TO 1 ( 9) DO LOOP ( 10) USER INTERACTION: Display Message: General Interactions Empty Waste Bottle. ( 11) DIGITAL IO: Read input from device 'Waste Sensor' into variable 'wastestate' ( 12) WHILE wastestate IS EQUAL TO 0 ( 13) DO LOOP ( 14) USER INTERACTION: Display Message: General Interactions Empty Vacuum Trap and Vacuum Condensate Trap. ( 15) DIGITAL IO: Read input from device 'Vacuum Trap Sensor' into variable 'vacstate' ( 16) WHILE vacstate IS EQUAL TO 1 ( 17) REMARK: ( 18) REMARK: BLOCK: Manual Setup ( 19) WIZARD Worktable Setup ( 20) USER INTERACTION: Display Message: General Interactions Fill both system liquid bottles with distilled water. Page 2

3 Empty vacuum trap and vacuum condensate trap. Empty waste container. ( 21) USER INTERACTION: Display Message: Rotor Setup Place 250ml Bottle Buffer A1 (with RNase A added) into Rotor Slot 1 250ml Bottle BufferA2 into Rotor Slot 3 500ml Bottle Buffer A4 (with ethanol added) into Rotor Slot 4 250ml Bottle Buffer A3 into Rotor Slot 5 500ml Bottle Buffer AW into Rotor Slot 6. DataDir\Bottle.bmp ( 22) USER INTERACTION: Display Message: Worktable Setup 1 Place a QIAplate Holder each into MP Slots 6, 11 and 16. <none> ( 23) USER INTERACTION: Display Message: Worktable Setup 2 Place NucleoSpin Filter 96 Plate into QIAplate Slot 6 and Seal unused wells. Place NucleoSpin Binding 96 Plate into QIAplate Slot 11. ( 24) USER INTERACTION: Display Message: Vac Setup Place Vac Filtration Adapter into QIAplate Slot 16. Place Vac Lid and Vac Top in the corresponding Vac Side Slot. ( 25) USER INTERACTION: Variable Input: Elution Volume Variable Name: ElutionVolume Default Parameters: No ( 26) EVALUATE TotalElutionVolume = ElutionVolume * NoSam * 1.2 / 1000 ( 27) USER INTERACTION: Display Message: Prepare Trough Rack Fill 12 ml Buffer AE into the disposable Trough in Therm Subslot A. DataDir\TroughRack.bmp ( 28) USER INTERACTION: Display Message: Elution Rack Page 3

4 Place a Microplate onto Elution Adapter into MP Slot 14. DataDir\Microtiter Plate.bmp ( 29) USER INTERACTION: Display Message: Sample Setup Place Flat-Bottom Block with samples into the Shaker back/left position. DataDir\Flat Bottom Block.bmp ( 30) END WIZARD ( 31) REMARK: ( 32) REMARK: BLOCK: Check Volume ( 33) FLUSH Probe to Use : All Probes Using Peristaltic Pump 30 ml Airgap After Flush : 0 Ask for More : yes ( 34) CHECK VOLUMES Check on Slot : Therm Subslot A Volume per Sample : yes Check One Fill All : yes Error on Less : yes Wash After Check : yes Airgap After Flush : 0 Alert Beep : yes Error on More : no Result on Error : yes Tolerance [ul] : 10 Offset [ul] : 0 Check the following positions: Position Volume [µl] Trough ElutionVolume ( 35) REMARK: BLOCK: Lysis ( 36) FLUSH DISPENSER Wash Volume [ml] : 30 ( 37) LOAD ROTOR Scan Rotor For Reagents : yes Check Expiration Date : no Check Volume : no Alert Beep : yes ( 38) FLUSH DISPENSER Wash Volume [ml] : 30 ( 39) ROBOT CONTROL: Call method 'CloseClamp' on device 'Shaker' ( 40) DISPENSE Reagent : BufferA1 Destination Slot : Shaker back/left Dispense Volume : 250 µl Prime Volume : 30 ml Wash Volume : 0 µl ( 41) START SHAKER Shake Duration : 04:00 Frequency : 1100 Interval : 30 ( 42) REMARK: Assembly of Vacuum Manifold Page 4

5 ( 43) MOVE RACK 'QIAplate Slot 16 Vac Base' Source Slot : QIAplate Slot 16 Destination Slot : Vac Base ( 44) MOVE RACK 'QIAplate Slot 11 Vac Base / Filtration Adapter' Source Slot : QIAplate Slot 11 Destination Slot : Vac Base / Filtration Adapter ( 45) MOVE RACK 'Vac Side Top Mount Vac Top Mount' Source Slot : Vac Side Top Mount Mount ( 46) MOVE RACK 'QIAplate Slot 6 Vac Top' Source Slot : QIAplate Slot 6 ( 47) REMARK: ( 48) FLUSH DISPENSER Wash Volume [ml] : 30 ( 49) WAIT until shaker finishes (Shaker) ( 50) START TIMER at 00:05:00. ( 51) DISPENSE Reagent : Buffer A2 Destination Slot : Shaker back/left Dispense Volume : 250 µl Prime Volume : 30 ml Wash Volume : 0 µl ( 52) START SHAKER Shake Duration : 01:00 Frequency : 600 Interval : 30 ( 53) FLUSH DISPENSER Wash Volume [ml] : 30 ( 54) WAIT until shaker finishes (Shaker) ( 55) WAIT until time has elapsed ( 56) DISPENSE Reagent : Buffer A3 Destination Slot : Shaker back/left Dispense Volume : 350 µl Prime Volume : 30 ml Page 5

6 Wash Volume : 0 µl ( 57) START SHAKER Shake Duration : 03:00 Frequency : 600 Interval : 30 ( 58) FLUSH DISPENSER Wash Volume [ml] : 30 ( 59) FLUSH Probe to Use : All Probes Using Peristaltic Pump 20 ml Airgap After Flush : 0 Ask for More : no ( 60) WAIT until shaker finishes (Shaker) ( 61) IF NoSam IS LESS THAN 96 ( 62) USER INTERACTION: Display Message: To Do... Penser a boucher les puits vides de la plaque filtrante avec un Tape Pad. DataDir\TurboFilter Plate.bmp ( 63) END IF ( 64) REMARK: ( 65) REMARK: BLOCK: Transfer and Filtration ( 66) EVALUATE SetIndexedPosVolumes(SettingVolumeTable, "Volume", "Shaker back/left") ( 67) TRANSFER Source Slot 0 : Shaker back/left Source Position 0 : Samples Aspiration Volume 0 : 1100 Dest Slot 0 : Vac Top Dest Position 0 : Samples Pos. Total Content : no Probe Select : All Steel Probes ( 68) START PUMP Pump Duration : 03:00 No Pressure Cont. : no Temperature : 60 Sample Binds to Membrane : Use Press Down Feature : Slot for Press Down Feature: Vac Top Using Vacuum Trap : Yes ( 69) FLUSH DISPENSER Wash Volume [ml] : 20 ( 70) FLUSH Probe to Use : All Probes Using Peristaltic Pump 10 ml Airgap After Flush : 0 Ask for More : no ( 71) ROBOT CONTROL: Call method 'OpenClamp' on device 'Shaker' ( 72) WAIT until vacuum pump finishes (RoboVac) ( 73) START TIMER at 00:00:05. Page 6

7 ( 74) WAIT until time has elapsed ( 75) ROBOT CONTROL: Call method 'Heat' on device 'RoboVac' ( 76) START TIMER at 00:00:15. ( 77) WAIT until time has elapsed (Timer) ( 78) REMARK: Move Vacuum Manifold ( 79) MOVE RACK 'Vac Top QIAplate Slot 6' Source Slot : Vac Top Destination Slot : QIAplate Slot 6 ( 80) MOVE RACK 'Vac Top Mount Vac Side Top Mount' Source Slot : Vac Top Mount Destination Slot : Vac Side Top Mount ( 81) MOVE RACK 'Vac Base / Filtration Adapter QIAplate Slot 16' Source Slot : Vac Base / Filtration Adapter Destination Slot : QIAplate Slot 16 ( 82) MOVE RACK 'Vac Side Top Mount Vac Top Mount' Source Slot : Vac Side Top Mount Mount ( 83) MOVE RACK 'QIAplate Slot 16 Vac Top' Source Slot : QIAplate Slot 16 ( 84) IF NoSam IS LESS THAN 96 ( 85) USER INTERACTION: Display Message: To Do... Penser a boucher les puits vides de la plaque filtrante d'elution avec un Tape Pad. DataDir\QIAprep Plate.bmp ( 86) END IF ( 87) REMARK: Page 7

8 ( 88) REMARK: BLOCK: Bind and Wash ( A4 et AW) ( 89) START TIMER at 00:00:02. ( 90) WAIT until time has elapsed (Timer) ( 91) START PUMP Pump Duration : 02:00 No Pressure Cont. : no Temperature : 60 Sample Binds to Membrane : Use Press Down Feature : Slot for Press Down Feature: Vac Top Using Vacuum Trap : Yes ( 92) WAIT until vacuum pump finishes (RoboVac) ( 93) START TIMER at 00:00:05. ( 94) WAIT until time has elapsed (Timer) ( 95) ROBOT CONTROL: Call method 'Heat' on device 'RoboVac' ( 96) DISPENSE Reagent : Buffer AW Dispense Volume : 600 µl Prime Volume : 30 ml Wash Volume : 0 µl ( 97) START PUMP Pump Duration : 02:00 No Pressure Cont. : yes Temperature : 60 Sample Binds to Membrane : Use Press Down Feature : Slot for Press Down Feature: Vac Top Using Vacuum Trap : Yes ( 98) WAIT until vacuum pump finishes (RoboVac) ( 99) START TIMER at 00:00:05. (100) WAIT until time has elapsed (Timer) (101) ROBOT CONTROL: Call method 'Heat' on device 'RoboVac' (102) DISPENSE Reagent : Buffer A4 Dispense Volume : 900 µl Prime Volume : 30 ml Wash Volume : 0 µl (103) START PUMP Pump Duration : 02:00 No Pressure Cont. : yes Temperature : 60 Sample Binds to Membrane : Use Press Down Feature : Page 8

9 Slot for Press Down Feature: Vac Top Using Vacuum Trap : Yes (104) WAIT until vacuum pump finishes (RoboVac) (105) START TIMER at 00:00:05. (106) WAIT until time has elapsed (Timer) (107) ROBOT CONTROL: Call method 'Heat' on device 'RoboVac' (108) DISPENSE Reagent : Buffer A4 Dispense Volume : 900 µl Prime Volume : 0 µl Wash Volume : 0 µl (109) START PUMP Pump Duration : 02:00 No Pressure Cont. : yes Temperature : 60 Sample Binds to Membrane : Use Press Down Feature : Slot for Press Down Feature: Vac Top Using Vacuum Trap : Yes (110) FLUSH DISPENSER Wash Volume [ml] : 30 (111) WAIT until vacuum pump finishes (RoboVac) (112) START TIMER at 00:00:05. (113) WAIT until time has elapsed (Timer) (114) ROBOT CONTROL: Call method 'Heat' on device 'RoboVac' (115) START TIMER at 00:00:15. (116) WAIT until time has elapsed (Timer) (117) REMARK: Move Vacuum Manifold (118) MOVE RACK 'Vac Top QIAplate Slot 12' Source Slot : Vac Top Destination Slot : QIAplate Slot 11 (119) MOVE RACK 'Vac Top Mount Vac Side Top Mount' Source Slot : Vac Top Mount Destination Slot : Vac Side Top Mount (120) MOVE RACK Alert Beep Silent Mode 'Vac Base QIAplate Slot 16' Source Slot : no : no : Vac Base Page 9

10 Destination Slot : QIAplate Slot 16 (121) MOVE RACK 'QIAplate Slot 12 Vac Base' Source Slot : QIAplate Slot 11 Destination Slot : Vac Base (122) MOVE RACK 'Vac Side Top Mount Vac Top Mount' Source Slot : Vac Side Top Mount Mount (123) MOVE RACK 'Vac Side Lid Mount Vac Lid Mount' Source Slot : Vac Side Lid Mount Destination Slot : Vac Lid Mount (124) REMARK: (125) REMARK: BLOCK: Dry plate (126) FOR DryCounter = 1 TO 4 WITH STEPSIZE 1 (127) START PUMP Pump Duration : 03:00 No Pressure Cont. : yes Temperature : 60 Sample Binds to Membrane : Use Press Down Feature : Slot for Press Down Feature: Using Vacuum Trap : No (128) WAIT until vacuum pump finishes (RoboVac) (129) START TIMER at 00:00:02. (130) WAIT until time has elapsed (Timer) (131) NEXT (132) START TIMER at 00:00:20. (133) WAIT until time has elapsed (Timer) (134) REMARK: Move Vacuum Manifold (135) MOVE RACK 'Vac Lid Mount Vac Side Lid Mount' Source Slot : Vac Lid Mount Destination Slot : Vac Side Lid Mount Page 10

11 Using LabHand Detect object : yes : yes (136) MOVE RACK 'Vac Top Mount Vac Side Top Mount' Source Slot : Vac Top Mount Destination Slot : Vac Side Top Mount (137) MOVE RACK 'Vac Base QIAplate Slot 12' Source Slot : Vac Base Destination Slot : QIAplate Slot 11 (138) MOVE RACK 'MP Slot 13 Vac Base' Source Slot : MP Slot 14 Destination Slot : Vac Base (139) MOVE RACK 'Vac Side Top Mount Vac Top Mount' Source Slot : Vac Side Top Mount Mount (140) MOVE RACK 'QIAplate Slot 12 Vac Top' Source Slot : QIAplate Slot 11 (141) REMARK: (142) REMARK: BLOCK: Elution (143) DISTRIBUTE Source Slot : Therm Subslot A Source Position : Trough Use Max. Syringe Volume : yes Dispense Volume : ElutionVolume Probe Select : All Steel Probes (144) START TIMER at 00:01:30. (145) FLUSH Probe to Use : All Probes Using Peristaltic Pump 10 ml Airgap After Flush : 0 Ask for More : no (146) WAIT until time has elapsed Page 11

12 (147) START PUMP Pump Duration : 01:30 No Pressure Cont. : yes Temperature : 23 Sample Binds to Membrane : Use Press Down Feature : Slot for Press Down Feature: Vac Top Using Vacuum Trap : Yes (148) WAIT until vacuum pump finishes (RoboVac) (149) START TIMER at 00:00:20. (150) WAIT until time has elapsed (151) MOVE RACK 'Vac Top QIAplate Slot 11' Source Slot : Vac Top Destination Slot : QIAplate Slot 11 (152) MOVE RACK 'Vac Top Mount Vac Side Top Mount' Source Slot : Vac Top Mount Destination Slot : Vac Side Top Mount (153) MOVE RACK 'Vac Base MP Slot 13' Source Slot : Vac Base Destination Slot : MP Slot 14 (154) REMARK: (155) REMARK: BLOCK: End of preparation (156) IF CHOICE IS 'yes' (157) EVALUATE ReportFile = "QIAprepTurbo 96 Report File " + GetCurrentDate("%d.%m.%Y") + ".csv" (158) PRINT REPORT on runtime to file Report of protocol run Date/Time: {GetCurrentDate()}, {GetCurrentTime()} Protocol name: {GetProtocolName()}.PRO Executor:???Please enter name here??? Page 12

13 Number of processed samples: 96 Number of run (today):???please enter number here??? Comments (159) END IF (160) USER INTERACTION: Display Message: Process done Process done! Close all buffer bottles. Stop Button: No <none> Page 13

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