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Placi PCR si qPCR FrameStar

Placile PCR FrameStar®

Tehnologia de realizare a placilor FrameStar® prin imbinarea a doua componente, reduce semnificativ gradul de evaporare din placile PCR, imbunatatind astfel rezultatele si la final un cost mai redus permitand utilizarea unui volum de reactie mai mic. 

 

Placile FrameStar® minimizeaza dilatarea si contractia in timpul proceselor PCR. Fiind formate din 2 componente, combina avantajele peretilor subtiri din polipropilena la nivelul godeurilor cu stabilitatea policarbonatului in timpul incalzirii si racirii la nivelul placii. Astfel evaporarea care apare in special la nivelul godeurilor din colt si randurilor externe este minima. De asemenea, lipsa deformari placi reduce variatiile semnalului fluorescent in cazul reactiilor qPCR.

 

Expansiunea termica a placilor din polipropilena determina evaporarea probelor mai ales de la nivelul godeurilor marginale.

Polipropilena reprezinta materialul optim pentru tuburile PCR deorece ofera cel mai eficient transfer termic precum si o suprafata inerta, cu capacitate de minima de legare pentru acizi nucleici, proteine si alte molecule. In ceea ce priveste constructia placilor polipropilena nu reprezinta cel mai bun material datorita faptului ca are un grad mare de dilatare si contractie in timpul fiecarui ciclu PCR (Fig.1). Miscarile de contactie si dilatare determina slabirea aderentei foliei si evaporarea continutului.  

Figure 1

Fig. 1: Placa standard din polipropilena sufera dilatari de pana la 2mm in timpul ciclulilor PCR, in special la nivelul godeurilor marginale.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Blocul termocyclerului nu impiedica aceasta dilatare a placilor PCR iar capacul incalzit accelereaza acest fenomen (Fig. 2).

 

 

Figure 2

Fig. 2: vedere laterala a unuei placi intr-un bloc PCR. Placa acoperita cu folie este prinsa intre termobloc si capacul incalzit dar singura parte fixa este la baza godeurilor.

 

 

 

 

 

 

 

 

 

 

 

 

 

Tehnologia cu 2 componente a placilor FrameStar® reduce dilatarea acestora si astfel evaporarea probelor. 

Marginile din policarbonat sunt mult mai rezistente termic comparativ cu cele din polipropilena ceea ce reduce la minim dilatarea. Din acest motiv foliile vor ramane fixate la nivelul godeurilor in timpul ciclurilor PCR. Pentru a compara rata de evaporare intre placile FrameStar si cele uzuale din polipropilena s-a adaugat cate 10µl  de apa in fiecare godeu al unei placi non-skirted din polipropilena si unei placi FrameStar cod catalog 4ti-0710. Ambele au fost acoperite cu folii qPCR cod catalog 4ti-0560. S-a cantarit greutatea ambelor placi inainte si dupa efectuarea unui program PCR (30 cicluri x 15'' 95C; 15'' 55C). Dupa cum se observa in tabelul 1, media volulmului evatorat din placile formate in intregime din polipropilena este de 2,3 µl/godeu, reprezentand 23% din volumul de reactie. Media volumului evaporat din placile FrameStar este de 0,49 µl/godeu.

  PCR (30 cycles x 15’’ 95°C; 15’’ 55°C).  Table 1 shows that the average volume loss from one piece PP plates was 2.3μl per well which is equal to 23% of the total reaction volume. in contrast the volume loss from FrameStar plates was only 0.49μl per well using adhesive sealing.
For easier sample identification we now supply all microplate designs with a printed alphanumeric grid reference.

 

Plate Type

Starting Weight

Weight post PCR

Weight loss

Volume loss total/well

FrameStar®4ti-0710

26.678g

26.631g

0.047g

47µl/0.49µl

One Piece

17.807g

17.586g

0.221g

221µl/2.3µl

Tab 1: Greutatea si volumul evaporat comparativ intre placile FrameStar si cele din polipropilena de 96 godeuri. rezultatele reprezinta o medie a 5 placi de fiecare tip. Dupa cum se observa din placile de polproplena se evapora un volum de 4 ori mai mare decat din placile FrameStar.

 

Evaporarea este mai intensa la nivelul godeurilor marginale.

In fig. 3 este ilustrat nivelul evaporarii in diferite zone ale placilor di polipropilena. Doar la nivelul celor 32 godeuri centrale se intalneste un nivel scazut de evaporare. La nivelul celor 2 randuri marginale, unde se gasesc 65% dintre godeuri evaporarea este intensa. 

Figure 3

 

Fig 3: Evaporarea atinge un nivel ridicat la nivelul godeurilor situate pe randul marginal (rosu), este medie la nivelul randului 2 (galben) si scazuta la nivelul godeurilor centrale (verzi)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2-component plates improve consistency of PCR results FrameStar®

We have compared the degree of evaporation from different areas of one piece PP and FrameStar® PCR plates. First, the 64 outer wells (two outer rows) of both plate types were filled with 10μl H2O, plates were then sealed with a qPCR adhesive (code 4ti-0560) and their total weight determined before and after PCR. The experiment was repeated with a set of plates of which the inner 32 wells (green area in Figure 3) were filled. Table 2 shows that evaporation from outer wells of standard PP plates was significant and 65% higher than from inner wells. in contrast, FrameStar plates showed low levels of sample loss across the plate.

 

Plate Type

Starting weight (g)

Weight post PCR (g)

Weight loss (g)

Volume loss total/well (g)

FrameStar® 4ti-0710, outer 64 wells

26.230

26.193

0.037

37µl/0.57µl

One piece, outer 64 wells

17.299

17.118

0.181

181µl/2.8µl

FrameStar® 4ti-0710, inner 32 wells

25.841

25.824

0.017

17µl/0.53µl

One piece, inner 32 wells

17.132

17.078

0.054

54µl/1.69µl

Table 2: Weight and volume loss from different sections of 96well PCR plates. Results shown are averages from plates of each plate type. Volume loss from the outer wells of one piece PP plates was 5-times higher than from FrameStar® plates.

 

The above results show that reaction volumes remain consistent across the 96wells (or 384wells, data not shown) in FrameStar® plates. in contrast, the reaction volumes in standard plates will differ increasingly between wells during PCR. Buffer concentrations in outer rows will increase dramatically and result in reduced enzyme activity. in extreme cases samples will fully evaporate.

 

FrameStar® 2-component technology allows for reduction of assay volumes and cost

Due to the much improved seal integrity, reaction volumes can often be reduced when using FrameStar® plates. Such downscaling of experiments can be successfully implemented without any loss of assay sensitivity or consistency (Figure 4) and reagent savings can be considerable.

Figure 4: Comparable Ct values and mean deviations were obtained with 15μl and 25μl SyBr Green assays in heat sealed FrameStar® plates.

Plate Distortion Post-PCR

Distortion tests have been performed with different PCR profiles using a variety of sealing materials. The results shown were obtained using the following protocol with 5 μl reaction volumes, plates sealed with adhesive sheets (4titude® code 4ti-0500), in a Thermo Px2 Cycler: 1min 94ºC, 30x(30sec 94ºC, 30sec 55ºC, 30sec 72ºC), 5min 72ºC, holding step at 4ºC. Averages from measurements of 10 plate samples are shown. X- and Y- axis measurements were taken in the middle sections of the plate skirt. Warpage was measured as a maximum deviation from a flat plane.

 

Plate Type

X-axis

Y-axis

Warpage

FrameStar® 96, skirted

0.02mm

0.02mm

0.03mm

One piece 96well PP Plate

1.18mm

1.69mm

1.80mm

FrameStar® 384

0.02mm

0.02mm

0.03mm

One piece 384well PP Plate

1.22mm

1.82mm

2.20mm

Table 3: Plate distortion post-PCR

 

Improved Sealing

In addition FrameStar® technology provides a much improved level of seal integrity: During PCR, with heat seals or adhesive seals, the excessive thermal expansion/contraction of standard one-piece polypropylene plates, can have a damaging effect on seal quality. This is especially evident around the edge of the plate where these forces are at their maximum resulting in significant sample evaporation from the outer wells. in contrast, the higher thermal stability of FrameStar® plates results in a reliable seal in all areas of the plate during PCR and in cold storage down to -80ºC.

 

Reduced Risk of Contamination

Raised rims around each tube aid the prevention of cross contamination between samples.

 

Plate Compatibility

FrameStar® plates are compatible with most PCR cyclers, real-time detection systems and sequencers.  The plates have been successfully tested for reliable use on most automation platforms.  Contact 4titude® for furth


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