TaqMan Residual DNA Detection Kit Chinese Hamster Ovary v2 Protocol

Transcription

1 TaqMan Residual DNA Detection Kit Chinese Hamster Ovary v2 Protocol Setting up the Real-Time PCR Reaction. Thaw the0x TaqMan Assay Mix Tube (0X TAM) from 20 C storage. Take out the 2X Environmental TaqMan Master Mix (2X EMM) from 4 C storage. Vortex each very gently tube before use and perform a quick spin. 2. It is recommended that you run the standards and unknown samples in triplicate. In addition, you should also run three no template controls on each plate. A no template control (NTC) has master mix, assay mix, and water, but no sample added to it. 3. It is also recommended that you run a total of 8 standard points of known concentrations in triplicate, preferably 0 fold dilutions. You can adjust the total number of standard points as you develop a standard operating protocol. 4. Standard samples should be CHO genomic DNA. The DNA should be as clean as possible and carefully quantitated, preferably by a fluorometer or a spectrometer. The recommended range of standard concentrations is between 00pg/ul to 0.0fg/ul in the final 30 µl reaction volume. This works out to 0.3 fg total genomic DNA in the lowest dilution standard sample (0.0fg/ul*30ul) and 3ng of total genomic DNA in the highest concentration standard (00pg/ul*30ul). 5. Add up to 0ul of the DNA extracted from unknown samples to each reaction. The other reaction components are the 2X Environmental master mix and the 0X TaqMan assay mix (contains two PCR primers and a FAM probe specific for CHO genomic DNA labeled as well as an internal positive control template, two PCR primers and VIC labeled probe for the internal positive control template). The total reaction volume is 30 ul, and the amount of unknown sample added to each well is 0ul (Table ). Water is used to bring the reaction mix to volume: Table : Reaction Mix Component Volume H 2 O 2 µl 2x Master Mix 5 µl 0x Assay 3 µl DNA Template 0 µl Total 30 µl 6. To determine how many reactions to prepare count the number of unknown samples and multiply by three, since they are run in triplicate, and allow for three no template controls. Count the number of standard samples and multiply by three since they are also run in triplicate. Add up the total number of reactions wells to be used, then mix at least 0% overage for pipetting loss. For example, if

2 you are running 8 standards and three unknowns, you ll have 8*3 standard points (24), 3*3 unknown samples (9), and 3 NTCs, for a total of 36 reaction wells. Prepare enough reaction mix for at least 40 reactions by multiplying all the volumes in the first column of table 2 by 40 (Table 2). Table 2: Preparing Master Mix for multiple reactions Component reaction 20 reactions 40 reactions 2X Master Mix 5.0 µl 300 µl 600 µl 0X Assay Mix 3.0 µl 60 µl 20 µl Water 2.0 µl 400 µl 80 µl 7. Pipet 20ul of the reaction mix into each well to be used on a plate. Add the 0 ul of standard sample, unknown sample, or 0 ul of water to the NTCs. Seal the plate with the optical film and continue with the Plate Set Up protocol (page 3). Set Up Example: A ng/μl B 00 C 0 D E 00 F 0 G H ng/μl ng/μl Sample Sample Sample 00 Sample Sample Sample Sample Sample Sample NTC NTC NTC 2

3 Setting up the reaction plate using the 7500 software. Double click on the software to launch. Click on Create New Document from the Quick Startup menu. If the Quick Startup menu doesn t appear click on the first icon in the menu bar at the top, the small white page. 2. In the New Document Wizard the assay type should remain on Standard Curve (Absolute Quantitation ), Container should remain on 96-Well Clear, and Template should be Blank Document. The Run Mode should be changed to Standard 7500 if it isn t on that setting all ready. In general the only setting you might have to change is the Run Mode; the other settings can remain on the default. If you are using a 7300 or a 7900 HT system please contact your field application specialist for information. 3. Click Next at the bottom of the Wizard page. 4. Select the CHO FAM detector from the list on the left and click Add in the middle of the screen. Select the VIC detector and click Add as well. You should see the two detectors in the list on the right side of the screen now. 5. Click Next at the bottom of the Wizard page. 6. Select the wells that will have sample in them by clicking and dragging along the wells, along the columns or row headers, or by holding down the control key and clicking on them individually. While they are selected click in the Use box in front of both of the detector names. You should now see the two detectors beside each other in each well you are using on the plate. If some wells to be used don t have the detectors in them just click on them and click the use box above. 7. Click Finish at the bottom of the page. 8. Now you need to specify which samples are standards, unknowns, and NTC. Double click in any well to launch the Well Inspector. Select the wells that are standards and then click in the Task box in the Well Inspector for the FAM CHO assay. You ll see a drop down menu appear. Change the task to Standard. The VIC detector can remain as Unknown. You should now see the detector boxes for the FAM CHO detector in the wells selected have S s in them instead of U s. Now highlight the replicates of the first standard dilution. Click in the Quantity box for the FAM CHO detector and type in the concentration of that standard, in numbers only (no units). If an error box appears when you first click in the quantity field just close it and continue. Fill in the concentration for all standard samples. You should see the numbers fill in beside the detector boxes for the FAM detector, and they should be in scientific notation. 9. Now select the no template control wells and click in the Task box. Change the task to NTC. Close the well inspector. 3

4 0. Highlight the replicates for your first unknown and type the name in. You ll see it fill in above the detector boxes in each well. Use the tab key or the arrow keys to move to the next set of replicates.. After sample set up is complete click on the Instrument tab in the upper left hand corner of the screen. 2. Change the sample volume to 30 ul. Make sure the Run Mode is on Standard Enter the following thermal cycling parameters Go to File and select Save and give the file a name and select where you want the file saved to on the computer, then hit Start. Analyzing the data After the run, the green arrow will be activated for analysis. The SDS software will automatically analyze the results and generate a standard curve. Concentration of the unknown samples will be automatically calculated against the standard curve. After the run is complete go to the Results tab and click on the Amplification Plot tab. Click on the top left hand corner of the plate in the bottom half of the screen to see the amplification plots. 2. Make sure the drop down menu beside Detector says All, then under Analysis Settings on the right hand side of the screen select Auto Ct then click the green analysis button in the top menu bar. 3. From the detector drop down menu select the VIC detector. Verify that all samples showed amplification of the VIC template, including the NTC wells. If a 4

5 well failed to amplify the VIC internal positive control that means PCR was inhibited in that well, and the sample should be repeated. 4. Go to the Standard Curve tab and look at the standard curve. Make sure the plate below is still selected. Look at the Standard Curve. Note the slope, this value should be consistent from run to run and should be around The absolute value is less important that the value being consistent, however, so note the slope from each run. The R2 value should be 0.99 or higher, if it is lower than that look at the standard curve and determine which points are not falling on the line. Highlight them, go to the Setup tab and omit them, then reanalyze. 5. Go to the report tab. The concentration of each unknown is reported here. If the sample says undetermined that means the well did not amplify and the amount of CHO residual DNA was below the sensitivity of the system. Verify all samples have a value for the VIC assay, again, this verifies that a sample could amplify and did not (validates a negative reaction is a true negative, not a failed reactions) Example of Real-time PCR results 5

6 Related Part Numbers for TaqMan Residual DNA Custom Detection Kit Chinese Hamster Ovary Consumables TaqMan Residual DNA Detection Kit Chinese Hamster Ovaryv2 P/N MagMAX -96 Viral RNA Isolation Kit P/N AM836 ABI PRISM Optical Adhesive Covers and 96-Well Optical Reaction Plates P/N Nuclease-free water P/N AM well regular PCR plate P/N ml Siliconized tubes to dilute DNA P/N AM2450 MicroAmp Optical Adhesive Film Kit P/N Instruments/Software: Applied Biosystems 7500 Real-Time PCR System P/N Applied Biosystems 7500 SDS software v.4 2CFRpartModule P/N or Applied Biosystems 7500 Fast Real-Time PCR System P/N Applied Biosystems 7500 Fast SDS software v.4 2CFRpartModule P/N or Applied Biosystems 7900 Fast Real-Time PCR System, 96-well Block P/N SDS Software 2.2 for the 7900HT System Enterprise Edition Upgrade Kit P/N