Accuracy & Precision of Liquid Transfer With Pins

 
 


MANUAL FP3 PIN REPLICATE TRANSFERS OVER TWENTY-FOUR
96 WELL PLATES

 
 


 Twenty-Four Plate Manual Replicator Transfers:  

Transferred to 200 ul 50 mM Tris, pH 8.0 in Assay Plate 1A

Pins washed in ~150 ml dH2O, blotted

Pins washed in ~150 ml Isopropanol, blotted, dried.

Pins transferred to 200 ul 50 mM Tris, pH 8.0 in Assay Plate 1B

Pins washed in fresh dH2O and Isopropanol, dried.

 

2.         Pins dipped into 200 ul FITC in 100% DMSO in Source Plate

Transferred to 200 ul 50 mM Tris, pH 8.0 in Assay Plate 2A

Pins washed in ~150 ml dH2O, blotted

Pins washed in ~150 ml Isopropanol, blotted, dried.

Pins transferred to 200 ul 50 mM Tris, pH 8.0 in Assay Plate 2B

Pins washed in fresh dH2O and Isopropanol, dried.

 

3-24.       Repeat through Assay Plates 24A and 24B

 

Add 1 ml 1M Tris, pH8.0 (final concentration ~50 mM Tris)

Aliquot 200 ul Tris/Wash to each well of black assay plate

Read Wash Plate

ASSAY OUTLINE:

     
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FITC Source Plate

Assay Plate           A

H2O  Wash           1

Isopropanol  Wash           1

Assay Plate           B

H2O  Wash           2

Isopropanol  Wash             2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Read after 24th plate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Mean FTIC Signal of Each Individual Pin Over Twenty-Four Manual Transfers

 
   

175199

167987

168780

179896

177477

172754

176475

178194

173510

180418

173092

175342

172809

166962

168435

171034

168005

168666

181265

169233

168903

173762

172993

169765

167298

165832

169438

175892

169829

178468

172546

174387

177447

171384

169894

169421

169084

167889

173436

181685

176511

170238

179931

179247

170418

173792

172997

179068

165882

176052

166423

168458

168968

174238

179874

173538

181658

179024

175375

169496

165960

173950

174790

182885

174083

175978

183721

181238

169725

181726

171937

167683

171445

167841

166939

164066

175782

170673

170084

178250

168350

177029

166365

171713

163360

170203

170746

164877

165561

166233

175879

168842

175449

172994

179367

180043

 
 


CV% of Each Individual Pin Over Twenty-Four Manual Transfers

 
   

7.0%

6.2%

6.5%

6.7%

7.1%

7.1%

6.8%

7.2%

7.4%

7.3%

8.2%

7.3%

5.7%

5.5%

5.3%

5.3%

6.2%

6.7%

7.1%

7.2%

6.8%

7.6%

7.6%

7.3%

6.5%

6.4%

4.7%

5.2%

5.8%

5.4%

6.4%

6.6%

6.6%

7.2%

7.3%

7.6%

7.4%

5.9%

4.9%

5.0%

5.9%

6.2%

5.9%

6.6%

6.2%

6.7%

7.3%

7.8%

6.2%

6.1%

5.2%

5.3%

6.2%

5.7%

6.5%

7.4%

6.6%

8.2%

7.3%

7.6%

7.5%

6.9%

6.0%

6.5%

6.4%

6.3%

6.0%

6.8%

6.7%

7.1%

7.0%

7.9%

6.7%

7.2%

6.3%

6.8%

6.5%

7.1%

6.6%

6.3%

6.0%

6.5%

7.0%

7.0%

7.3%

7.9%

7.2%

7.9%

7.4%

6.3%

8.4%

7.6%

7.3%

7.5%

6.4%

7.6%

 
   

12210

10367

10889

12027

12556

12204

12077

12797

12838

13240

14214

12837

9870

9101

8968

9045

10444

11369

12941

12231

11566

13185

13072

12412

10879

10534

7916

9149

9823

9579

11002

11583

11641

12318

12417

12836

12507

9917

8424

9013

10445

10474

10695

11756

10618

11671

12609

14009

10316

10692

8672

8943

10516

9939

11731

12823

11959

14614

12736

12803

12427

11942

10489

11892

11121

11015

10979

12379

11437

12977

12022

13289

11469

12009

10463

11143

11483

12090

11245

11206

10163

11498

11609

11974

11928

13380

12297

13092

12249

10459

14758

12768

12807

12946

11533

13598

 
   

All Plates

Std Dev.

12429

Mean

172832

Maximum

216038

Minimum

140955

CV%

7.2%

mg FITC/well

0.0293

nl FITC Transferred

73.299

     
 
 

FP3 Manual Transfer from 200 ul FITC in 100% DMSO to 200 ul 100 mM Tris

Plate

Maximum

Minimum

SD

Mean

CV%

nl FITC

Plates 1-24

216038

140955

12429

172832

7.2%

73.30

1

207644

143009

9719

171663

5.7%

72.80

2

211517

167070

9650

184928

5.2%

78.43

3

212178

180009

7194

193870

3.7%

82.22

4

204834

166483

7572

183722

4.1%

77.92

5

187296

154746

6995

170794

4.1%

72.43

6

190975

161041

6120

175591

3.5%

74.47

7

186223

150395

6439

165857

3.9%

70.34

8

180768

152809

5740

164568

3.5%

69.79

9

190867

156054

6714

175488

3.8%

74.43

10

186579

159381

5258

170918

3.1%

72.49

11

191800

160959

6567

175268

3.7%

74.33

12

176449

142058

6642

161516

4.1%

68.50

13

188681

160106

6293

175448

3.6%

74.41

14

184977

152484

6684

165178

4.0%

70.05

15

196107

149651

8128

173638

4.7%

73.64

16

178699

153365

5701

164942

3.5%

69.95

17

199595

157176

7212

181541

4.0%

76.99

18

179882

151136

6206

164982

3.8%

69.97

19

216038

150650

15251

173344

8.8%

73.52

20

179822

149277

6981

162970

4.3%

69.11

21

192684

156311

8472

175750

4.8%

74.54

22

177233

140955

7181

157268

4.6%

66.70

23

179045

147937

6155

163005

3.8%

69.13

24

215578

165308

13099

195711

6.7%

83.00

25

430151

303965

25364

357447

7.1%

151.62

 
 


NOTE:  Plate 25 – Pin tool was removed from source liquid very quickly, unlike the other 24 plates where the pin tool was removed from the source liquid as slowly as possible.  The CV% is still within an acceptable range (<10%), but the volume of liquid transferred nearly doubled.
Assay Plate 1A –First assay plate, FITC signal

 
   

169954

160741

163572

165667

164413

167288

171099

176075

172041

179844

177524

182328

162323

161294

158151

165263

158023

156790

164097

163181

163977

169880

175720

169038

166274

143009

161827

165656

161331

163463

166094

167650

172973

166381

168697

175345

185950

166779

171058

176807

165530

168716

166263

165339

159300

169171

171629

186721

148176

176898

163972

167727

170549

174030

174859

155179

179395

207644

175241

169876

168523

182243

183752

188768

181105

171246

178152

170113

170535

171022

173451

173415

167016

177541

180895

176586

182576

171472

162691

176134

166016

178764

162286

177531

175060

186317

185781

180315

173922

176930

191206

173033

172198

174624

194064

190573

Max

207644

Min

143009

Std Dev

9719

Mean

171663

CV%

5.7%

 
 
Assay Plate 24A – Last assay plate, FITC signal
     
   

181298

180601

178768

198900

196016

192528

205291

208601

205606

214667

209647

209211

184482

180525

182936

186540

190970

194940

215578

203157

202086

210387

213019

207711

173716

180560

183467

197983

194892

201692

204829

208296

213967

209924

212326

208443

174634

181335

181897

194180

203371

195199

209259

214197

200669

208470

213106

215049

170276

185561

181626

184850

195253

200044

206111

204730

213119

208850

212518

202622

174774

187383

184373

202668

195209

196420

202688

208545

197783

211880

206879

195114

180749

165308

178431

174324

190362

191287

196759

200220

195025

203776

197976

203254

170937

175710

182521

176706

177407

171405

191624

189288

199363

197849

205592

208221

Max

215578

 

Min

165308

 

Std Dev

13099

 

Mean

195711

 

CV%

6.7%

 

 
 
Assay Plate 1B first test for contamination of wash solution, FITC signal
     
 

96

54

54

61

74

45

59

44

40

29

33

51

59

59

84

51

63

52

154

47

57

69

43

51

180

95

52

39

54

52

56

67

93

62

52

41

146

52

39

50

52

48

46

78

59

112

62

51

83

55

51

70

49

36

58

64

51

63

42

41

52

56

56

55

54

94

56

51

56

83

55

39

93

78

31

83

52

36

60

45

49

53

107

42

82

50

68

172

39

47

61

45

33

80

29

44

Max

180

 

Min

29

 

Std Dev

27

 

Mean

62

 

CV%

44.3%

 

 
   

71

69

40

71

57

51

51

40

37

43

35

35

39

42

57

43

42

63

58

46

46

57

36

33

69

51

57

47

42

43

33

58

39

39

38

29

76

53

40

74

42

39

45

47

49

50

37

43

70

40

52

64

49

57

41

51

60

54

42

33

48

41

76

41

49

43

49

52

44

67

36

35

43

52

28

55

40

45

37

42

49

39

37

39

44

55

53

61

51

65

40

41

40

56

30

39

Max

76

 

Min

28

 

Std Dev

11

 

Mean

48

 

CV%

23.4%

 

 
 
(Each well represents ~0.13% of the total wash reservoir after 24 transfers) FITC signal
 
   

1308

1312

1298

1258

1321

1415

1251

1347

1305

1349

1270

1377

1200

1236

1270

1357

1255

1446

1331

1318

1263

1377

1299

1281

1318

1365

1336

1389

1313

1476

1340

1317

1350

1291

1289

1321

1352

1380

1348

1341

1327

1424

1399

1371

1375

1394

1238

1419

1368

1310

1392

1374

1328

1514

1460

1322

1339

1410

1235

1399

1298

1317

1395

1363

1328

1456

1351

1369

1308

1339

1417

1366

1357

1388

1381

1342

1402

1434

1406

1383

1353

1395

1409

1308

1424

1256

1428

1293

1381

1423

1384

1289

1470

1319

1420

1350

 
 


Conclusion

     
 


These experiments demonstrate that the 2X wash and blot steps are sufficient to clean the pins and that the wash solution does not get grossly contaminated even after 25 wash and blot steps.  Both accuracy and precision data were better than 9% CV in a manual assay.

 

  Graphs Showing Maximum and Minimum Signal Values For Each Plate
  Across The 24 Plates
 
 

 


  Graphs Showing Mean Signal Values and CV% For Each Plate
  Across The 24 Plates

 
 
 

     

  Graphs Showing Average Volume Transferred (nl) For Each Plate
  Across The 24 Plates
 
 
     
         

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