FrameStar. 2-component plates & standard PCR plastic

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Nathan Goodman
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1 FrameStar 2-component plates & standard PCR plastic

2 When optimising PCR experiments or establishing new technologies the attention is always focused on 1.Instrumentation 2.Reagents

3 Important: Choose the best consumables! The design of plates and choice of sealing are equally important factors which will influence the quality of results. The right choice will save money!!

4 1. PCR Plastic Production standard Design of plates Choice of materials (plates and sealing) all are important factors which influence the quality of the result.

Production Standard Precision tooling and

5 Production Standard Precision tooling and moulding Virgin, medical grade polymers Clean-room moulding ISO certified production Multi-level QC Visual & biological testing. DNase/RNase, human DNA and Pyrogen Performance testing. PCR leak test 100% inspection of products

6 Quality Control Using an electrical discharge method we test every plate for well integrity eliminating the possibility of holes or knit lines that could lead to sample loss.

7 Tube Design The wall thickness of tubes determines thermal transfer rate Thinner tubes are less of a heat barrier, samples reach temperature quicker which improves the result But thinner walls are more difficult to manufacture

8 Types of PCR Plates Polycarbonate (PC) plates (one piece) Polypropylene (PP) plates (one piece) 2-Component plates PC frame PP tubes

9 Polypropylene vs. Polycarbonate Polycarbonatebest for rigidity of plate Substantially less thermal expansion than pp but not recommended for PCR tubes (porous) Polypropylenebest for PCR tubes Inert,low DNA & protein binding,flexible but too soft to give plate rigidity

10 One Piece Polypropylene Plates We offer skirted, non skirted and semi-skirted options High quality & price competitive Top seller because people don t know how to sell FrameStar

Thermal Expansion of PP Plates Standard plates with polypropylene frame expand by up to 2mm during thermal cycling The co-efficient of expansion of the seal is

11 Thermal Expansion of PP Plates Standard plates with polypropylene frame expand by up to 2mm during thermal cycling The co-efficient of expansion of the seal is different from the plate 55 C 95 C Movement of wells will weaken the seal and lead to evaporation in corner positions and outside rows when using sealing sheets

12 Evaporation from Polypropylene PCR Plates Evaporation from outside wells is the strongest!

13 Evaporation has a detrimental effect on PCR A 20% loss in volume leads to a ph change of 1 at 74 C Reduced enzyme efficiency when evaporation occurs Differential evaporation from wells in a PCR plate makes quantification difficult or impossible!

14 One piece PP plates also distort post- PCR making them more difficult to handle

15 FrameStar 2-Component Plates Polypropylenetubesfor optimum PCR performance Polycarbonate frame gives the plate stability Much reduced thermal expansion and contraction Much reduced evaporation Improved PCR performance Reaction volumes can be reduced (savings!) At least EVERYBODY doing qpcr should use 2-component plates!

16 Volume loss in one-piece and FrameStar plates Plate Type FrameStar 4ti-0710 outer 64 wells FrameStar 4ti-0710 Inner 32 wells One-piece 4ti-0750 outer 64 wells One-piece 4ti-0750 inner 32 wells Starting volume 10µl Volume Loss per well 0.57µl 0.53µl 2.80µl 1.69µl

17 Sealing Comparison FrameStar 4ti-0960 vs One-Piece Skirted 96 4ti-0740 Type of Plate Weld Seal (4ti-0575) qpcr Seal (4ti-0560) Flat caps (4ti-0751) % volume loss / loss per well One piece PP 4ti-0740 Framestar 96 4ti % /1.15µL 13.9% / 3.48µL 6.5% / 1.63µL 3.0% / 0.75µL 4.9% /1.22µL 4.3% / 1.08µL Based on a 30 cycle PCR of 1min at 95 C and 1 min at 55ºC using a 25uL reaction volume.

18 Best Plate/Seal vs Worst Best: Framestar/weld seal 3.0%=0.75µL/well Worst: 4ti-0740/qPCR seal 13.9%=3.48µL/well A 2.73µL/well evaporation reduction representing approx US$ 27 saving per 96 well plate. Based on US$0.10 per µl

with cloned cdna target: Red PP, Blue Framestar

19 Identical seal, differing plate design Identical human GAPDH ThermoFisher Solaris probes and primers with cloned cdna target: Red PP, Blue Framestar Visibly improved Cq values Improvement in PCR efficiency

20 Other FrameStar Benefits 4titude currently has 10 different plate designs covering the widest range of compatible thermal cyclers. Eppendorf and Biorad are the only other licensed suppliers of 2 component PCR plates and only direct competitors US Patents 7,347,977 and 6,340,589 Both have a much smaller range, only supporting their own instruments 4titude offers 2 component plates at prices which are normally charged for standard one-piece polypropylene plates! Colour coding (6 colours) and white well tubes. Have had requests for black tubes PFIA Printed alpha numeric grid reference.

21 FrameStar -White Tube Plates For quantitative PCR and other fluorescence applications. White wells to reflect the light created during the reaction and pigment chosen low in autofluorescence. Compared to clear and frosted wells: Higher sensitivity Lower levels of fluorescence detectable Higher consistency (less well to well variation) Black plate deck for reduced background. Some designs available with clear frames for easier labelling

22 How do we sell FrameStar? Identify if you are selling against one-piece PP plates or 2-component plates. i.e. Eppendorf and Biorad If One-Piece PP Plates: Establish if sample leakage has been a customer problem. Especially in corner wells!! Less repeats. FrameStar OFFERS A SOLUTION Establish if customers are having plate handling issues due to plate distortion. i.e. stackers, robotic arms, dispensing. FrameStar OFFERS A SOLUTION Establish if reagent saving is of interest! FrameStar OFFERS A SOLUTION

23 FrameStar Key Summary Warp much less during PCR compared with standard plates Beneficial for robotics use (more reliable with automated pipetting and stacking) Less thermal expansion and shrinkage for improved sealing integrity! Less sample evaporation, especially from corner wells Beneficial for low volume PCR The 4titude FrameStar plates are licensed by Eppendorf. US Patents 7,347,977 and 6,340,589

24 This is what our customers think

25 1.Miniaturisation of SyBrGreen Assays Laboratory of Dr. Carolin Deiner, FU Berlin Work performed on BioradI-Cycler using heat sealed FrameStar PCR plates. The aim of the work was to reduce reagent volumes to reduce costs without compromising on the quality of the results

26 Identical Ct Values were obtained with 15μl and 25μl SyBr Green Assays in heat sealed FrameStar plates qpcr graph of Ubiquitin, data provided by Dr. Carolin Deiner, FU Berlin

27 Reducing the SyBr Green consumption by 40% means considerable cost savings to us Dr Caroline Deiner SyBr Green cost of 96x 25μl reactions: USD 77 SyBr Green cost of 96x 15μl reactions: USD 46 Saving per plate: USD 31

28 2. Miniaturisation of TaqmanAssays Dr.Andreas Dahl, Max-Planck Institute f. Molecular Genetics Berlin, Department of Prof. Hans Lehrach Work performed on Roche LightCycler480 using standard & FrameStar PCR plates with adhesive & heat seals

29 Dr. Dahl s group works on assay miniaturisation down to nanoliter levels The aim of the work described here was to identify the minimum reaction volumes for Taqman assays in 384well plates

30 Sealing Efficiencies with standard and FrameStar PCR Plates Heat Sealing vs Adhesive Sealing % of wells w. detectable liquid Heat Sealing Adhesive Sealing nl Standard PCR Plate FrameStar 2-Component Plate The use of heat sealing and/or FrameStar plates reduces the evaporation during PCR

31 Cp Values TaqMan RNase P detection kit on the Roche LightCycler LC480 in FrameStar 384well plates Volume Template Conc n (RNase P) Cp Average CP Median STDEV CV Dropouts 0.5µl 1: % 0% 1µl 1: % 0% 2µl 1: % 0% 3µl 1: % 0% Variation in Cp values is low and comparable to 10μl reactions *Note: Automated nano-dispenser was used for those experiments

32 Conclusions FrameStar plates offer real benefits over one piece polypropylene PCR plates. Evaporation can be minimised by choosing the correct PCR plate and seal. Substantial cost savings can be made when reducing reaction volumes without compromising on the quality of results. The correct choice of PCR consumable will improve your customers PCR results.

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