Applied Biosystems 7900 HT (for SDS Software 2.1, 2.3, and 2.4) instrument setup instructions for EpiTect Methyl II PCR Arrays

Transcription

1 Prtcl Sheet Applied Bisystems 7900 HT (fr SDS Sftware 2.1, 2.3, and 2.4) instrument setup instructins fr EpiTect Methyl II PCR Arrays Imprtant pints befre starting Please read the handbk supplied with the EpiTect Methyl II PCR Array, paying careful attentin t the Safety Infrmatin and Imprtant Ntes sectins, befre beginning this prcedure. Please make sure the system has been prperly calibrated fr the regin f interest (ROI), backgrund, and pure dye. (Refer t the manufacturer s Installatin and Maintenance manual fr the calibratin prcedures if needed). Please make nte f the installed SDS Sftware versin n yur instrument. Prcedure Creatin f PCR prtcl template 1. Open the ABI 7900 Fast System SDS Sftware n the desktp f the cmputer that is cnnected t the ABI 7900HT system. 2. Select File New ; the New Dcument dialg bx will appear. Select Assay SDS 2.1: Abslute Quantificatin (Standard Curve) SDS 2.3 : Standard Curve (AQ) SDS 2.4 : Standard Curve (AQ) Select Cntainer SDS 2.1: 384-well Clear Plate SDS 2.3: 96 r 384 wells Clear Plate SDS 2.4: 96 r 384 wells Clear Plate Select Template SDS 2.1: Blank Dcument SDS 2.3: Blank Template SDS 2.4: Blank Template The field fr Barcde is ptinal. Click OK. Under the Setup tab n the center-right, select Add Detectr buttn frm bttm. Sample & Assay Technlgies

2 In the Detectr Manager windw: SDS2.1 Select SYBR fr the reprter dye. Click Cpy T Plate Dcument buttn. Click Dne buttn (See Figure 1). Chse ROX fr the Passive Reference bx. SDS2.3 Click New In the Add Detectr dialg bx: Enter SYBR in the Name field. Select SYBR fr the Reprter. Click OK. Click nce n the newly added Detectr. Click Cpy T Plate Dcument buttn. Click Dne buttn. Chse ROX fr the Passive Reference bx. SDS2.4 Click New In the Add Detectr dialg bx: Enter SYBR in the Name field. Keep Grup field as Default. Keep Descriptin and AIF Assay ID empty. Select SYBR fr the Reprter. Select Nn Flurescent fr Quencher. Click the square with clr beside Clr, then select preferred clr in the Select Detectr Clr/Swatches page (usually green fr SYBR). Click OK t clse the page. Click OK. Click nce n the newly added Detectr. Click Cpy T Plate Dcument buttn. Click Dne buttn. Chse ROX fr the Passive Reference bx. ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

3 SYBR ROX Figure 1. Plate setup selecting dye. Select the Instrument tab, then the Thermal Prfile tab. Highlight Stage 1 (50 C fr 2:00 minutes) by right-clicking the muse and dragging it acrss Stage 1. Delete Stage 1 by clicking the Delete buttn. Then cntinue t edit the Thermal Prfile as fllws: Enter 95.0 C fr 10:00 minutes fr Stage 1 ; fr Stage 2, enter 99.0 C fr 0:15 (15 secnds) fllwed by 72.0 C fr 1:00 minute with Repeats 3 (3 cycles). Fr Stage 3, enter 97.0 C fr 00:30 secnds fllwed by 72.0 C fr 1:00 minute with Repeats 40 (40 cycles). Click the Add Dissciatin Stage buttn; the pre-set dissciatin stage will be added as Stage 4. Fr SDS Sftware 2.1: Uncheck the bx next t 9600 Emulatin. Fr SDS Sftware 2.3/2.4: select Standard mde. Put 10 µl fr Sample Vlume. T set up the data cllectin cnditins (see Figure 3), select the Data Cllectin tab, then left-click s that the Data Cllectin ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

4 symbl is belw the 72 C segment in Stage 3, the cnnecting line between the 60 C and 95 C segments in Stage 4, and the 95 C segment in in Stage 4. Remve the remaining Data Cllectin symbls assciated with all ther temperatures and cnnecting lines by left-clicking. Then select File Save As t save the template file. Save the file as an SDS 7900HT Template Dcument (*.sdt) with the filename EpiTect Methyl II PCR Array Prtcl 7900HT Template (click Save ). Alternatively, dwnlad the PCR prtcl template file (EpiTect Methyl II PCR Array Prtcl Template-ABI7900.sdt) frm the SABisciences website ( Figure 2. Plate setup setting thermal prfile. ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

5 Data cllectin symbl Figure 3. Plate setup setting data cllectin. Perfrming real-time PCR detectin 3. If the thermcycler is ff, press the pwer buttn t switch n the instrument. Wait fr the instrument t bt and display the Pwer status light. Switch n the cmputer cnnected t the thermcycler. 4. Make sure that there are n bubbles in any well, and that the reactin mix is psitined at the bttm f each well. If nt, centrifuge the plate at ~1000 x g fr 60 secnds. 5. Open the ABI 7900HT Fast System SDS Sftware. 6. Select File New. In the New Dcument Wizard dialg bx, select Brwse t lad the EpiTect Methyl II PCR Array Template file. Then click Finish. This will lad the previusly saved setup t the new plate dcument. T set up a sample plate (See Figure 4), click the square buttn in the upper left crner f the full plate layut panel (between the letter A and 1 ) t select all wells. Once selected, the lwer ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

6 full plate table panel will be highlighted in yellw and the bx next t SYBR n the left panel will be checked. 7. Save the new dcument under a new filename as SDS 7900HT Dcuments (*sds). 8. Select the Instrument/Real-Time tab. Click Cnnect/Cnnect t Instrument t cnnect the cmputer t the thermal cycler. Click Open t pen the plate tray and place yur plate in the precisin plate hlder with A1 in the tp left crner. Click Clse t lad the plate. Click Start/Start Run t begin the PCR run. Wait 30 secnds t 1 minute fr the initial priming, then the run shuld start. The estimated run time will then appear n the screen. Buttn t select all wells SYBR Figure 4. Plate setup selecting sample. After the PCR run 9. When the PCR run is cmplete, a small dialg bx stating The run cmpleted successfully will appear n the screen. Click OK ; this will clse the bx. 10. Select Analysis Analysis Settings ; the Analysis Settings dialg bx will appear. Select the radi buttns next t Autmatic Baseline and Manual C T (0.2 is usually fine; please refer t step 13 fr manual selectin f the threshld), and click OK. 11. Click the Result tab. Display Data as ΔRn vs. Cycle. If all wells are nt yet selected, click the square buttn in the upper left crner f the diagram f the plate (between the letter A and 1 ) t select all wells. The selected wells will be highlighted in yellw in the lwer left panel. ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

7 12. Select Analysis Analyze. The sftware will calculate the data and generate the C T (lwer-left panel). 13. Fllw the prcedures belw t select the baseline and threshld manually (see figures 5 and 6): Nte: Please use the same threshld fr all yur plates in ne study. T define the baseline, use the Linear View f the amplificatin plts. Duble-click n the Y-axis. The windw fr Display Settings will appear. Fr Amplificatin Plt Prperties, select Aut Scale fr bth the Y and X-Axes. Select Linear View fr Y-Axis, then click OK. With the linear plts, determine the cycle number at which the earliest amplificatin can be seen. Use the red sliding bars n the X-axis t set the manual baseline t start frm cycle number 2 thrugh tw cycle values befre the earliest visible amplificatin. T define the threshld value, use the Lg View f the amplificatin plts. Duble-click n the Y-axis. The windw fr Display Settings will appear. Fr Amplificatin Plt Prperties, select Aut Scale fr bth the Y and X-Axes. Select Lg View fr Y-Axis and click OK. With the lg plts, place the threshld line abve the backgrund signal but within the lwer third f the linear phase f the amplificatin plt. Select all samples Linear view Set the baseline Figure 4. Setting the baseline. ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

8 Select all samples Lg view Set the threshld Linear phase C T values Figure 5. Setting the threshld. The values fr C T will be displayed in the lwer left panel fr each well. T exprt the result t an Excel spreadsheet, select File Exprt Results Table and save the file as a Tab-delimited Text file (*txt). This file frmat can be pened in Micrsft Excel. ABI 7900 real-time PCR setup instructins fr EpiTect Methyl II PCR Arrays

9 The EpiTect Methyl II PCR Arrays are intended fr mlecular bilgy applicatins. These prducts are nt intended fr the diagnsis, preventin, r treatment f a disease. Fr up-t-date licensing and prduct-specific disclaimers, see the respective QIAGEN kit handbk r user manual. QIAGEN handbks can be requested frm QIAGEN Technical Service r yur lcal QIAGEN distributr. Selected handbks can be dwnladed frm Safety data sheets (SDS) fr any QIAGEN prduct can be dwnladed frm Trademarks: EpiTect, QIAGEN (QIAGEN Grup); Excel, Micrsft (Micrsft Crpratin); Applied Bisystems, SYBR (Life Technlgies Crpratin). Oct QIAGEN, all rights reserved. France The Netherlands Australia Austria 0800/ Belgium Canada China Denmark Finland Germany Hng Kng Ireland Italy Japan Krea (Suth) Luxemburg Nrway Singapre Spain Sweden Switzerland UK USA Sample & Assay Technlgies