kvantifikace proteinu mg/ml prumer 0 0.0710 0.0670 0.0690 0.125 0.0720 0.0830 0.0775 0.25 0.0860 0.0920 0.0890 0.5 0.1120 0.1050 0.1085 1 0.1520 0.1500 0.1510 2 0.2110 0.2240 0.2175 "na 0,4mg/ml" C prumer mg/ml prepocet bfb+merc T- 0.1180 0.1320 0.1250 0.729333333 65.87 14.59 T-W+ 0.1370 0.1250 0.1310 0.809333333 81.87 16.19 T-R+ 0.1150 0.1140 0.1145 0.589333333 37.87 11.79 T-W+R+ 0.1150 0.1160 0.1155 0.602666667 40.53 12.05 D T+ 0.1310 0.1380 0.1345 0.686842105 57.37 13.74 T+W+ 0.1190 0.1190 0.1190 0.482894737 16.58 9.66 T+R+ 0.1240 0.1110 0.1175 0.463157895 12.63 9.26 T+W+R+ 0.1070 0.1190 0.1130 0.403947368 0.79 8.08 ug/ml 0 0.0760 0.0870 0.0815 0.125 0.0840 0.0870 0.0855 0.25 0.1060 0.1070 0.1065 0.5 0.1200 0.1270 0.1235 1 0.1640 0.1460 0.1550 2 0.2460 0.2300 0.2380 4 0.3700 0.4000 0.3850 CD hodnoceni ucinnosti transfekce - GFP protein CD Plot 1 (FSC-A/SSC-A) Plot 2 (FL1-A) P1 P2 M1 M2 Count Count Count % of This Plot Count % of This Plot A01 CD T- 361 "3,815" "3,185" 98.67% 4 0.12% A02 CD T+ 957 "6,645" "5,411" 95.67% 31 0.55% A03 CD T-W+ 449 "3,237" "2,605" 96.98% 8 0.30% A04 CD T+ W+ 357 "1,216" 955 97.35% 6 0.61% A05 CD T-R+ 373 "2,590" "2,040" 96.36% 16 0.76% A06 CD T+R+ 282 922 722 95.25% 10 1.32% A07 CD T-W+R+ 228 "1,524" "1,247" 96.74% 11 0.85% A08 CD T+W+R+ 826 "1,178" 882 92.94% 18 1.90% prumerna ucinnost 0.80% AUTOFLUORESCENCE koncentrace RNA "C,D" konc. RNA 800ng/ul doredit na H2O D T+ 68.85 11.6194626 D T+R+ 98.8 8.097165992 3.402834008 D T+W+ 102.25 7.82396088 3.67603912 D T+W+R+ 94.05 8.506113769 2.993886231 [RNA} T- C 143.5 5.574912892 5.925087108 [RNA} T- W+C 118.45 6.753904601 4.746095399 [RNA} T- R+C 84.9 9.422850412 2.077149588 [RNA} T- W+R+C 94.8 8.438818565 3.061181435 Top flash FF R pomer FF/R x100 T- 297 37460 0.007928457 0.792845702 T-W+ 211 12504 0.0168746 1.687460013 T-R+ 257 14282 0.017994679 1.799467862 T-W+R+ 3268 22274 0.146718147 14.67181467 T+ 294 44996 0.006533914 0.653391413 T+W+ 587 98452 0.005962296 0.596229635 T+R+ 537 39622 0.013553077 1.355307657 T+W+R+ 4893 72630 0.067368856 6.736885584 transfekce a následný top flash presne odpovídají očekávání!!! GAPDH Pos Name Cp prumer A1 T- 24.92 24.505 A2 T- 24.09 "brat nebudeme, " B1 T-W+ 24.06 24.015 nebyl tam dost specificky vzorek B2 T-W+ 23.97 C1 T-R+ 23.83 23.86 C2 T-R+ 23.89 D1 T-W+R+ 23.14 23.17 D2 T-W+R+ 23.2 E1 T+ 23.12 23.13 E2 T+ 23.14 F1 T+R+ 37.77 32.005 F2 T+R+ 26.24 G1 T+W+ 23.4 23.65 G2 T+W+ 23.9 H1 T+R+W+ 24.79 25.415 H2 T+R+W+ 26.04 HPRT Pos Name Cp A3 T- 24.98 24.87 A4 T- 24.76 B3 T-W+ 25.3 25.075 do pomeru bereme GAPDH B4 T-W+ 24.85 C3 T-R+ 24.62 24.53 C4 T-R+ 24.44 D3 T-W+R+ 24.3 24.3 D4 T-W+R+ 24.3 E3 T+ 23.79 23.825 E4 T+ 23.86 F3 T+R+ 25.53 25.5 F4 T+R+ 25.47 G3 T+W+ 24.09 24.14 G4 T+W+ 24.19 H3 T+R+W+ 24.24 24.17 H4 T+R+W+ 24.1 C-MYC "cely divny,dvojband" Pos Name Cp prumer Cp HPRT DCp 2^-DCp x1000 A5 T- 35.16 34.985 24.87 10.115 0.000901741 0.090174054 A6 T- 34.81 B5 T-W+ 34.78 35.99 25.075 10.915 0.000517914 0.051791394 B6 T-W+ 37.2 C5 T-R+ 32.81 32.985 24.53 8.455 0.002849649 0.284964911 C6 T-R+ 33.16 D5 T-W+R+ 34.63 34.465 24.3 10.165 0.000871024 0.087102394 D6 T-W+R+ 34.3 E5 T+ 31.58 31.25 23.825 7.425 0.005819053 0.581905259 E6 T+ 30.92 F5 T+R+ 31.29 31.115 25.5 5.615 0.020404059 2.040405917 F6 T+R+ 30.94 G5 T+W+ 31.05 31.02 24.14 6.88 0.008490116 0.849011611 G6 T+W+ 30.99 H5 T+R+W+ 31.05 31.05 24.17 6.88 0.008490116 0.849011611 AXIN2 Pos Name Cp A7 T- 25.61 25.565 24.87 0.695 0.617709319 61.77093186 A8 T- 25.52 B7 T-W+ 25.46 25.58 25.075 0.505 0.704660378 70.46603776 B8 T-W+ 25.7 C7 T-R+ 25.1 25.11 24.53 0.58 0.668963777 66.89637774 C8 T-R+ 25.12 D7 T-W+R+ 24.79 24.835 24.3 0.535 0.690158677 69.01586767 D8 T-W+R+ 24.88 E7 T+ 24.58 24.535 23.825 0.71 0.611320139 61.13201388 E8 T+ 24.49 F7 T+R+ 25.86 25.835 25.5 0.335 0.792784137 79.27841366 F8 T+R+ 25.81 G7 T+W+ 24.66 24.675 24.14 0.535 0.690158677 69.01586767 G8 T+W+ 24.69 H7 T+R+W+ 24.7 24.735 24.17 0.565 0.675955417 67.59554165 H8 T+R+W+ 24.77 CYCLIN D1 Pos Name Cp A9 T- 26.43 26.355 24.87 1.485 0.357248535 35.72485349 A10 T- 26.28 B9 T-W+ 26.59 26.565 25.075 1.49 0.356012549 35.60125489 B10 T-W+ 26.54 C9 T-R+ 26.5 26.46 24.53 1.93 0.262429171 26.24291709 C10 T-R+ 26.42 D9 T-W+R+ 26.45 26.445 24.3 2.145 0.226094844 22.60948444 D10 T-W+R+ 26.44 E9 T+ 26.31 26.24 23.825 2.415 0.187504874 18.75048737 E10 T+ 26.17 F9 T+R+ 26.47 26.23 25.5 0.73 0.602903914 60.29039138 F10 T+R+ 25.99 G9 T+W+ 26.5 26.59 24.14 2.45 0.183010712 18.3010712 G10 T+W+ 26.68 H9 T+R+W+ 26.66 26.615 24.17 2.445 0.183646079 18.36460792 H10 T+R+W+ 26.57 RNF43 Pos Name Cp A11 T- 25.37 25.335 24.87 0.465 0.724471077 72.44710773 A12 T- 25.3 B11 T-W+ 24.97 25.06 25.075 -0.015 1.010451446 101.0451446 B12 T-W+ 25.15 C11 T-R+ 24.91 24.92 24.53 0.39 0.763129604 76.31296045 C12 T-R+ 24.93 D11 T-W+R+ 24.26 24.27 24.3 -0.03 1.021012126 102.1012126 D12 T-W+R+ 24.28 E11 T+ 24.02 24.02 23.825 0.195 0.873572896 87.35728959 E12 T+ 24.02 F11 T+R+ 25.5 -25.5 47453132.81 4745313281 F12 T+R+ G11 T+W+ 24.31 24.29 24.14 0.15 0.901250463 90.12504626 G12 T+W+ 24.27 H11 T+R+W+ 24.41 24.38 24.17 0.21 0.864537231 86.45372313 H12 T+R+W+ 24.35 cMyc Axin2 cyclinD1 RNF43 T- 0.09 61.77 35.72 72.45 T-W+ 0.05 70.47 35.60 101.05 T-R+ 0.28 66.90 26.24 76.31 T-W+R+ 0.09 69.02 22.61 102.10 T+ 0.58 61.13 18.75 87.36 T+R+ 2.04 79.28 60.29 T+W+ 0.85 69.02 18.30 90.13 T+R+W+ 0.84 67.60 18.36 86.45 divnz nevychazi nevychazi "nevyslo, hodnoty T+" jsou mal0 Western blotting "hezky vysel RNF43, pLRP6, trosku tušíme aktivní beta catenin. Disheveled by mel mit dva bandy," aktin a tubulin jsou asi spatna protilatka… ##### Sheet/List 2 ##### kvantifikace proteinu mg/ml prumer 0 0.0710 0.0770 0.0740 0.125 0.0780 0.0790 0.0785 0.25 0.0980 0.0990 0.0985 0.5 0.1150 0.1180 0.1165 1 0.1450 0.1890 0.1670 2 0.2640 0.2770 0.2705 4 0.4010 0.4010 prepocet bfb A A mg/ml "na 0,5 mg/ml" T- 0.1240 0.1150 0.1195 0.497619048 0 8 T-W+ 0.1510 0.1150 0.1330 0.658333333 25.33333333 10.53333333 T-R+ 0.1440 0.1300 0.1370 0.705952381 32.95238095 11.2952381 T-W+R+ 0.1090 0.1240 0.1165 0.461904762 0 8 32.3 B B T+ 0.1110 0.1070 0.1090 2.082352941 253.1764706 33.31764706 T+W+ 0.0840 0.0840 0.0840 1.101960784 96.31372549 17.63137255 T+R+ 0.0790 0.0840 0.0815 1.003921569 80.62745098 16.0627451 T+W+R+ 0.0810 0.0770 0.0790 0.905882353 64.94117647 14.49411765 mg/ml 0 0.0520 0.0530 0.0525 0.125 0.0600 0.0620 0.0610 0.25 0.0630 0.0620 0.0625 0.5 0.0670 0.0700 0.0685 1 0.0830 0.0830 0.0830 2 0.1070 0.1080 0.1075 4 0.1520 0.1620 0.1570 kvantifikace RNA Sample ID ng/ul A260 A280 260/280 T- 28.69 0.717 0.417 Jan-72 T- 24.99 0.625 0.315 Jan-98 T- 21.91 0.548 0.293 Jan-87 T+ 78.81 1.970 0.966 2-Apr T+ 78.17 1.954 0.969 2-Feb T- W+ 62.21 1.555 0.761 2-Apr T- W+ 62.67 1.567 0.758 2-Jul T+W+ 22.55 0.564 0.314 Jan-80 T+W+ 21.41 0.535 0.265 2-Feb T-R+ 176.09 4.402 2.207 2.00 T-R+ 199.04 4.976 2.492 2.00 T+R+ Aug-13 0.203 0.082 Feb-47 T+R+ Sep-28 0.232 0.123 Jan-88 T+R+ Sep-45 0.236 0.114 2-Aug T-R+W+ 207.60 5.190 2.605 Jan-99 T-R+W+ 203.08 5.077 2.592 Jan-96 T+R+W+ 95.21 2.380 1.182 2-Jan T+R+W+ 100.98 2.524 1.284 Jan-97 T-R+ 30x 49.83 1.246 0.652 Jan-91 T-R+ 30x 49.09 1.227 0.649 Jan-89 T-R+W+ 30x 52.21 1.305 0.697 Jan-87 T-R+W+ 30x 51.36 1.284 0.681 Jan-89 kvantifikace RNA ng/ul prumer do realu 1000 ng "doredit na 11,5" T- 28.69 24.99 26.84 268.4000 3.73 7.77 T+ 78.81 78.17 78.49 784.9000 1.27 10.23 T-W+ 62.21 62.67 62.44 624.4000 1.60 9.90 T+W+ 22.55 21.41 21.98 219.8000 4.55 6.95 T-R+ 49.83 49.09 49.46 1483.8000 0.67 10.83 T+R+ 9.28 9.45 9.37 93.6500 10.68 0.82 T-W+R+ 52.21 51.36 51.79 1553.5500 0.64 10.86 T+W+R+ 95.21 100.98 98.10 980.9500 1.02 10.48 Topflash B T+ T+ W+ T+ R+ T+R+W+ TF 143 141 151 5403 Ren 1722 1093 7356 2587 result 0.083042973 0.129002745 0.020527461 2.088519521 A T- T- W+ T- R+ T-R+W+ TF 182 150 130 113 Ren 341 351 331 335 result 0.53372434 0.427350427 0.392749245 0.337313433 T- T- W+ T- R+ T-R+W+ T+ T+ W+ T+ R+ T+R+W+ 0.53372434 0.427350427 0.392749245 0.337313433 0.083042973 0.129002745 0.020527461 2.088519521 ucinnost transfekce Plot 1 (FSC-A/SSC-A) Plot 2 (FL1-A) P1 P2 M1 M2 Count Count Count % of This Plot Count % of This Plot B01 AB T- 238 "3,508" "3,209" 98.16% 45 1.38% B02 AB T+ 241 "4,121" "3,655" 94.35% 170 4.39% B03 AB T-W+ 189 "3,838" "3,525" 97.38% 75 2.07% B04 AB T+ W+ 407 "4,503" "3,981" 92.80% 190 4.43% B05 AB T-R+ 147 "2,365" "2,108" 97.05% 33 1.52% B06 AB T+R+ 260 "2,126" "1,689" 90.08% 114 6.08% B07 AB T-W+R+ 118 "2,491" "2,098" 95.41% 50 2.27% B08 AB T+W+R+ 127 "3,804" "3,135" 91.67% 144 4.21% vyhodnoceni qRT PCR GAPDH Pos Name Cp A1 T+ 13.86 14.52 A2 T+ 15.18 B1 T+ W+ 24.6 24.61 B2 T+ W+ 24.62 C1 T+ R+ 25.31 25.255 C2 T+ R+ 25.2 D1 T+ W+ R+ 15.58 15.455 D2 T+ W+ R+ 15.33 E1 T- 19.12 19.065 E2 T- 19.01 F1 T- w+ 17.11 17.205 F2 T- w+ 17.3 G1 T- R+ 15.94 15.595 G2 T- R+ 15.25 H1 T- W+ R+ 14.81 14.765 H2 T- W+ R+ 14.72 HPRT Pos Name Cp A3 T+ 19.45 19.46 A4 T+ 19.47 B3 T+ W+ 25.77 25.735 B4 T+ W+ 25.7 C3 T+ R+ 26.75 26.83 C4 T+ R+ 26.91 D3 T+ W+ R+ 19.63 19.53 D4 T+ W+ R+ 19.43 E3 T- 23.17 26.245 E4 T- 29.32 F3 T- w+ 20.52 20.575 F4 T- w+ 20.63 G3 T- R+ 19.66 19.6 G4 T- R+ 19.54 H3 T- W+ R+ 18.57 18.66 H4 T- W+ R+ 18.75 C MYC Pos Name Cp prumer Cp HPRT DCp 2^-DCp x1000 A5 T+ 27.77 28.175 19.46 8.715 0.002379708 2.379707546 A6 T+ 28.58 B5 T+ W+ 25.735 B6 T+ W+ 35 C5 T+ R+ 35 26.83 C6 T+ R+ 35 D5 T+ W+ R+ 27.57 27.525 19.53 7.995 0.003919812 3.919811518 D6 T+ W+ R+ 27.48 E5 T- 35 26.245 E6 T- 35 F5 T- w+ 29.21 29.4 20.575 8.825 0.002205009 2.205008603 F6 T- w+ 29.59 G5 T- R+ 27.33 27.38 19.6 7.78 0.004549741 4.549740572 G6 T- R+ 27.43 H5 T- W+ R+ 26.9 26.965 18.66 8.305 0.003161884 3.161883658 H6 T- W+ R+ 27.03 AXIN 2 Pos Name Cp A7 T+ 27.62 27.455 19.46 7.995 0.003919812 3.919811518 A8 T+ 27.29 B7 T+ W+ 26.88 26.935 25.735 1.2 0.435275282 435.2752816 B8 T+ W+ 26.99 C7 T+ R+ 27.64 27.365 26.83 0.535 0.690158677 690.1586767 C8 T+ R+ 27.09 D7 T+ W+ R+ 27.24 27.285 19.53 7.755 0.004629269 4.629268636 D8 T+ W+ R+ 27.33 E7 T- 26.85 26.69 26.245 0.445 0.734584317 734.5843166 E8 T- 26.53 F7 T- w+ 27.92 27.99 20.575 7.415 0.005859527 5.859527302 F8 T- w+ 28.06 G7 T- R+ 27.73 27.795 19.6 8.195 0.003412394 3.412394125 G8 T- R+ 27.86 H7 T- W+ R+ 28.55 28.38 18.66 9.72 0.001185737 1.185737192 H8 T- W+ R+ 28.21 CYCLIN D1 Pos Name Cp A9 T+ 24.64 24.64 19.46 5.18 0.027584469 27.58446863 A10 T+ 24.64 B9 T+ W+ 27.24 B10 T+ W+ 26.98 C9 T+ R+ 27.3 C10 T+ R+ 27.76 D9 T+ W+ R+ 24.15 24.125 19.53 4.595 0.041377778 41.37777845 D10 T+ W+ R+ 24.1 E9 T- 27.07 27.04 26.245 0.795 0.576343173 576.3431734 E10 T- 27.01 F9 T- w+ 25.23 25.24 20.575 4.665 0.039418044 39.41804403 F10 T- w+ 25.25 G9 T- R+ 23.71 23.845 19.6 4.245 0.052738487 52.73848726 G10 T- R+ 23.98 H9 T- W+ R+ 23.94 24.915 18.66 6.255 0.013093549 13.09354898 H10 T- W+ R+ 25.89 RNF43 Pos Name Cp A11 T+ 27.55 28.7 19.46 9.24 0.001653799 1.653799438 A12 T+ 29.85 B11 T+ W+ 26.52 26.61 25.735 0.875 0.545253866 545.2538663 B12 T+ W+ 26.7 C11 T+ R+ 26.63 26.865 26.83 0.035 0.976031761 976.0317608 C12 T+ R+ 27.1 D11 T+ W+ R+ 26.88 26.88 19.53 7.35 0.006129563 6.129563265 D12 T+ W+ R+ E11 T- 26.22 26.37 26.245 0.125 0.917004043 917.0040432 E12 T- 26.52 F11 T- w+ 27.53 27.545 20.575 6.97 0.007976657 7.976657232 F12 T- w+ 27.56 G11 T- R+ 27.55 27.49 19.6 7.89 0.004215735 4.215735299 G12 T- R+ 27.43 H11 T- W+ R+ 26.91 26.83 18.66 8.17 0.003472042 3.472041723 H12 T- W+ R+ 26.75 c Myc Axin2 cyclin D1 RNF43 T+ 2.38 3.92 27.58 1.65 T+W+ 435.28 545.25 T+R+ 690.16 976.03 T+W+R+ 3.92 4.63 41.38 6.13 T- 734.58 576.34 917.00 T-W+ 2.21 5.86 39.42 7.98 T-R+ 4.55 3.41 52.74 4.22 T-W+R+ 3.16 1.19 13.09 3.47 upravy dle RNF 43 c Myc Axin2 cyclin D1 RNF43 T+ 2.379707546 3.919811518 27.58446863 1.653799438 T+W+ 435.2752816 545.2538663 T+R+ 690.1586767 976.0317608 T+W+R+ 734.5843166 576.3431734 917.0040432 T- 3.919811518 4.629268636 41.37777845 6.129563265 T-W+ 2.205008603 5.859527302 39.41804403 7.976657232 T-R+ 4.549740572 3.412394125 52.73848726 4.215735299 T-W+R+ 3.161883658 1.185737192 13.09354898 3.472041723 "bohuzel doslo asi k prehozeni vzorku (podle RNF43), ale celkove to vyslo podivne, i když " technicky je to oK Western blotting "hezky vysel pLRP6, RNF43 a aktivní bcatenin, to vše dle očekávání, ostatní byla asi nevhodná protilátka"