Patricia Koutz Director of R&D

1. Introduction to V&P Scientific Pin Tools and Accessories for Tecan Workstations Patricia Koutz Director of R&D

2. Advantages of V&P Pin Tools Delivery from 2 nl to 5 ul. Nanoliter delivery with CVs averaging ~5% fluorescein. Convert a retired 96 robot to 384 or 1536 pin tool station. Accommodates 1536 format easily. Nanoliter delivery facilitates assay miniaturization. Pin tool assay procedure shortens cycle time by half.

3. Advantages of V&P Pin Tools Eliminates dilution step in screening assays to save time, reagents and disposable costs. (10-50nL direct into assay plate) No disposable tip costs – pays for tool by second run. Method development is quick. Pin tools mount to 45 commercial robot systems. Pin tools are useful in many applications.

4. Tecan Floating Pin Tool – MCA/TeMO

5. Tecan Floating Pin Tool – RoMA

6. What Makes a Pin Tool? • The Pins • The Fixture- 2 float plates • The Mounting Plate

7. V&P Pins Slot pin with liquid in slot Solid pin with hanging drop Large selection of pin diameters, slots, lengths, and coatings to accommodate many applications. Slot width is typically less than 1/3 the diameter of the pin. 7

8. V&P Pin Diameters

9. Tube & E-Clip Style Pins

10. E-Clip Style Pins 1.58mm 1.58mm Common Uses • Larger Volume Transfers (500nl-5ul) • Cell Transfers (Yeast, E. coli, etc.): • Liquid-to-liquid (Solid or slotted pins) • Liquid-to-membrane (Solid or slotted pins) • Liquid-to-agar (Solid pins only) • Agar-to-agar (Solid pins only) 1.58mm 1.58mm 4.77mm 2.36mm 2.36mm 3.18mm

11. FP6 Slot Pins 1.58mm diameter

12. Tube-Style Pins Common Uses • Very Small Volume Transfers (2-500nl) • Compound Transfers

13. FP1 Slot Pins

14. FP3 Slot Pins

15. Misc. Info • Liquid transfers with slotted pins • Slots fill by capillary action • Slots do not need to be submerged to fill • Slots MUST be submerged to transfer liquid • The slot of the FP6S5 pin (5ul volume) is 6mm deep. • Watch liquid displacement in wells of microplate as pins enter the wells.

16. V&P Floating Pin Tool Fixture

17. V&P Mono Pin Tool Fixture Mono Pin Tool on MWG RoboAmp 17

18. V&P Rotational Mounting Plate for Tecan MCA/TeMO Pin Tools

19. V&P Mounting Plate for Tecan RoMA Pin Tools

20. Guide for Selectingthe Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Non-specific binding to stainless steel. “Z” clearance on robot deck.

21. Guide for Selectingthe Right Pin Tool • Robot system. • V&P RoMA Mounting Plate (BMPROMA) • V&P standard MCA/TeMO mounting plate (BMPTECAN) • V&P rotational MCA/TeMO mounting plate (BMPTECANR) • Volume to be transferred. • Volume in source plate. • Solid pins versus slotted pins. • Non-specific binding to stainless steel. • “Z” clearance on robot deck.

22. Tecan TeMO Pin Tool(Yale)

23. Tecan RoMA Pin Tool (U of Kentucky)

24. V&P Docking Station for Tecan Pin Tools 24

25. V&P Docking Station for Tecan Pin Tools 25

26. Guide for Selectingthe Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Non-specific binding to stainless steel. “Z” clearance on robot deck.

27. Factors Affecting Pin Delivery Volume Pin diameter. Volume of slot in pin. Surface tension of the liquid being transferred. Surface tension of the pin. – how clean? Uneven transfers due to the sides and slot volume variation Depth to which the pin is submerged in source plate. SPEED OF REMOVAL OF PIN FROM SOURCE LIQUID.

28. Speed of removal of pins • Pull pins out SLOW from source plates • Excess liquid will wick off and LESS volume will be transferred • Pull pins out FAST • Excess liquid does not wick off and HIGHER volumes will be transferred • INCREASE SPEED = INCREASE VOLUME

29. Speed of removal of pins During Customer Set Up: • Customers set up a standard curve and mock fluorescein assay at various withdrawal speeds to determine the appropriate speed for the volume desired. • CV’s generally do not change with speed.

30. V&P Scientific Nanoliter Pins 1 Volume range determined by speed of withdrawal from source plate 2 Change in speed 7-fold, liquid transferred in DMSO

31. What happens exactly? • Pin tools are a TRANSFER tool, not a dispensing tool. • Example: If you transfer 5 ul from the source plate to 45 ul in the destination plate with a pin tool, you will have 50 ul of a 1:10 dilution in the destination plate…while the pins are in the wells. • When the pins are withdrawn from the wells, they will remove 5 ul of the 1:10 dilution, leaving 45 ul final volume in the destination plate. • Solid pins transfer liquid on the sides of the pins and larger hanging droplet • Slotted pins transfer liquid on the sides, in the slot and hanging droplet

32. What happens exactly? • Pre-wet pins in source plate • Move pins up and down through the meniscus, 3X (completely out of the liquid). • If the pins hit a air bubble going into well, this mixing will displace it. • Similar to pre wetting pipetting tips. • Post-transfer mix in destination plate • Move pins up and down through the meniscus, 3X. • Pins act as piston moving up and down to mix. • Just letting pins sit in liquid doesn’t help increase amount of volume transfer.

33. Last Withdrawal Speed • The LAST mix withdrawal of the pins from the source plate is the most important. • This speed is determined from the mock fluorescein assay run during set up. • Use the same speed and number of cycles in source and destination plates so the transfer amounts will be the same. • The first two up and down mix movements can be faster.

34. Guide for Selectingthe Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Non-specific binding to stainless steel. “Z” clearance on robot deck. Solid pins versus slotted pins.

35. Affect of Variable Source Plate Volumes on Pin Transfer Volume

36. Guide for Selectingthe Right Pin Tool • Robot system. • Volume to be transferred. • Volume in source plate. • Solid pins versus slotted pins. • Cost, CV’s • Non-specific binding to stainless steel. • “Z” clearance on robot deck.

37. Hydrophobic/Lipophobic Coating Prevents or reduces non-specific binding. Prevents or reduces lipid and lipoprotein binding. Prevents liquid “creep” up the pin. Increases size of hanging drop. Decreases volume on side of pin. Coating can be applied to all V&P stainless steel pins. At least $2 more vs. uncoated pin. Whole pin coated including slot.

38. Guide for Selectingthe Right Pin Tool • Robot system. • Volume to be transferred. • Volume in source plate. • Solid pins versus slotted pins. • Non-specific binding to stainless steel. • “Z” clearance on robot deck. • Long pins versus short pins

39. Tube-Style Exposed Pin Length

40. Questions To Ask Customers • What is the pin tool format? • 96, 384 or 1536 pins? • What are the mother and daughter plate formats? • 96, 384 or 1536? • Standard microplates or deep well plates? • What is the typical volume in the mother and daughter plates? • For example: • Average of 50ul liquid in the 384 well mother plate • Average of 200ul liquid in the 96 well daughter plate or agar surface in Nunc Omni Tray for daughter plate. 40

41. Questions To Ask Customers(Cont.) • What volume do you need to transfer between the mother and daughter plate? • What type of sample is being transferred? • For example: • Transferring compound libraries in 100% DMSO from mother plate to aqueous cell-based assays in daughter plates. • Transferring bacterial or yeast cells in culture media from mother plate to agar surface in daughter plate. • Will the pin tool be attached to the instrument head or be picked up with the gripper arm? 41

42. Pin Tool Data

43. Are your pins clean?

44. Pin Validation Assay(Fluorescein Transfer)Materials • V&P Scientific pin tools. • CyBi™-Well 96 Liquid Handling Robot. • Fluorescein Isothiocyanate (FITC), Isomer 1. Sigma P/N F7250. • 100% DMSO. Sigma P/N D2650. • 100 mM Tris, pH 8.0. Sigma P/N T3038. • Microfluor 96-Well Microtiter Plates, Dynex Technologies P/N 7805. • PerkinElmer/Wallac VICTOR2 Multilabel Reader.

45. Pin Validation AssayMethods • Dip pin tool into source plate (3X) • Pins removed from source plate at controlled speeds • Dip pin tool into assay plate (3X) • Wash pins • Repeat • Withdrawal speeds: 0.78-5.76 cm/sec

47. Pin Tool Maintenance • Daily cleaning – Before and after assay. • Cleaning in-between sample transfers. • Heavy duty cleaning. • Frequently used pin tools - clean once a week • Infrequently used pin tools - clean once a month

48. VP 540DB Blotting Station

49. VP 110 Pin Cleaning Solution • Available in 30ml bottles of 5X concentrated solution. • Dilute 1:5 in distilled water. • Dilution can be reused up to two months if stored in sealed container.

50. Cleaning Pins In-BetweenSample Transfers (cont.) • Thorough washing uses 7 deck positions total • 96 & 384 pin tools can be blotted in offset positions in VP 540DB Blotting Station. • ~2.25-4.5mm offset. • Blot in same position on Blotting Station for each wash station. • Watch how high the pins are pushed up into the float plate during blotting. • If excess liquid is not blotted from slots, the pins may take 60-90 sec to dry unless a fan or vacuum system is used.