Bio Rad PCR Pictorial Guide 111011 - UF/IFAS

Guide to using the Bio RadCFX96 Real‐Time PCRMachineKyle Dobbs and Peter Hansen

Table of Contents Overview .3Setup Reaction Guidelines 4Starting up the Software 5Setup Protocol on Software 6Setup Plate on Software 9Standard Curve Setup .14Setup Plate 16Start .18Data Analysis of Output .19Fold Change Calculations 222

Overview This pictorial guide to using the Bio‐Radreal‐time PCR machine is meant tosupplement the Polymerase Chain Reactionguide that is also on Dr. Peter /Protocols/PCR protocol%20Fear%20et%20al.pdf). Screen shots andpictures contained in this guide are moreinformational purposes only. For moreinformation, please see the instructionmanual located on the desktop computerfor this machine. In general, we suggest that you setup youplate on the computer using excel firstfollowed by setting up the plate on the Bio‐Rad computer software before actuallypipetting anything. ALWAYS USE GLOVES WHEN TOUCHINGTHE REAL‐TIME PCR MACHINE3

Setup Reaction Guidelines We recommend using excel or some other spreadsheetsoftware to setup your real time plate and master‐mixreaction amounts prior to using the machine. This willmake the setup and your analysis easier. We use a total reaction volume of 20 μl per well. This includes atotal master‐mix amount of 18.8 μl (6.8 μl of water, 1 μl of .5 μMforward primer, 1 μl of .5 μM reverse primer and 10 μl of SsoFastEvaGreen Supermix with Low ROX (Bio‐Rad)). We then add 1.2 μlof cDNA sample to each of their respective wells.When setting up the plate, we always duplicate each sample toallow for averaging the cycle threshold value that is generated tocompensate for pipetting error.Included a negative control from your reverse transcriptionprocedure and a water sample.4

Starting up the software The Real‐time PCR machine has itsown dedicated desktop computerthat is currently running Microsoft7. Login information and passwordare located on the top of thecomputer. Also at this time, turnon the CFX96 machine next to thecomputer. Double‐click on the icon labeled Bio‐Rad CFX Manager. Login to the software. Ifyou do not have a user IDplease see Luciano Bonillaor Kyle Dobbs5

Setup Protocol on theSoftware When first starting the software, it willprompt you to create a new file,repeat a file or do other things thatwill be discussed later. Click on“Create a new file.” After clicking on “Create anew file”, you will need tocreate the PCR program thatthe machine will use.Initially, the software using adefault program, but you willneed to change this in orderto use the real‐timecapabilities of the machine.After doing this once, you willnot need to do this step againfor future PCR reactions.6

Setup Protocol on theSoftware To change the program protocol, click onthe “Edit Selected” button in the top leftof the window You will need to change theprogram to what is picturedon the right.7

Setup Protocol on theSoftware Once the program has been changed, click OK. The softwarewill ask for to save the file, do this as you can use this againby clicking on “Select Existing” the next time you need to runa Real‐time PCR. You should now have ascreen that looks exactlylike the picture on theright. You are now readyto proceed to the platesetup8

Setup Plate on Software Using the Excel spreadsheet you created earlier, you will nowinsert the same information into the software. *Note: Thesoftware is not as “intuitive” as Excel, i.e., you cannot copy and paste, etc. After clicking on the“Plate” tab locatednext to the“Protocol”, the imageon the left shouldappear. This is thedefault plate setupthat the softwaregives you. We willchange this to mirrorwhat you have onyour Excelspreadsheet. Click on the “Edit Selected”button in the top right of thewindow displayed above.You will then see the windowdisplayed to the right. Asmentioned before, this is thedefault that the softwaregives you.9

Setup Plate on Software Highlight all of the wells andclick on the clear wellsbutton. All of the wells are now clearof the default settings. We use Sybr green with low ROX as our Master‐mix,and we therefore only use one fluorophore. Youcan click on the “Select Fluorophore” button anddeselect all fluorophores except Sybr. The softwaredoes not care which fluorophore you select so couldjust as easily select ROX, but we only need one.10

Setup Plate on Software Now, you can begin thereplication of your Excel fileonto the software. Firsthighlight the area on the platewhere you are going to beplacing all of your samples. Once highlighted, you can now select what those wellsare to become. This is done by clicking on the “SelectType” button on the left. Scroll down until you find whattype of sample you are inserting.11

Setup Plate on Software Repeat the previous step for thewater samples on your plate byhighlighting those wells andselecting “NTC.” Repeat the previous step forthe water samples on yourplate by highlighting thosenegative control from thereverse transcription reactionand selecting “Neg.” Next, highlight all of the wells that are using the same primerfor analysis and click on the button located to the rightlabeled “Target Name.” *If your gene name or “Target” is not in thescroll down list, type your gene of interest into the box.12

Setup Plate on Software Repeat this step for your different “Targets” until you aredone. Once done, click the “OK” button and save the platesetup.13

Standard Curve Setup(skip if already determined) If you are using new primers, you need to determine theirefficiency. More information on this is located on thePolymerase Chain Reaction protocol online while this portionis how set‐up those wells on the Bio‐Rad machine. Highlight the wellsyou want to usefor standard curveanalysis and clickon Standard underthe “Sample Type”button. Next, set the replicate size by clicking onthe “Replicate Series” button. Assign theappropriate replicates14

Standard Curve Setup(skip if already determined) Next, assign your dilution values by firstentering a starting concentration.Usually we use our nanodrop reading.The starting concentration is going tobecome your first standard or in thisinstance, well A1. You can also changeyour dilution factor from 10 to whateverdilution value you used. Normally, wemake A1 the highest concentration,therefore A2‐A5 decrease. You should see something similar to the picture below.15

Setup Plate To see the reaction setup, please see the “Setup ReactionGuidelines” page. When using the Bio‐Rad machine, you canONLY use Bio‐Rad plates, master‐mix and plate covers.Failure to do so will result in undesired results. Plates andplate covers are located in the top shelf of the molecularbiology bench Bio‐Rad 96‐well plates Bio‐Rad 96‐well plateclear covers EvaFast Master‐Mix with low ROX is located in two places.The currently used Master‐Mix is located in walk‐in fridge inthe “Molecular Biology” drawer while the stock tubes arelocated in the ‐20 freezer located in the lab. Bio‐RadMaster‐Mix Location ofStock Location of 16currently used

Setup Plate When making the master‐mix for the wells, it is crucial to useDEPC treated water. We have a stock bottle located to theright of the molecular biology bench. PLEASE aliquot a smallamount ( 3‐4 ml) into a 10 ml tube and label this with yourinitials. ONLY use your tube and do not share your tube withothers. Be extremely careful not to contaminate the stock. When making your plate, use a new aerosol tip each timeyou pipette something. Once you are finished with your plate, you need to place acover on the top of the plate. Be extremely careful not toshake or bump you plate while doing this. You can centrifugethe plate at this step if needed. We use the blunt end of asharpie to seal the gaps between the wells. Failure to do sowill allow the master‐mix in each well (along with yoursample) to evaporate during the real‐time process.Top rightcornershould A117

Start! After closing the lid on the Bio‐Rad machine, the “Start Run”button will appear and the software is ready to start the run.After clicking the “Start Run” button, you will be prompted tosave the data file. This will save the data generated by therun at each of the steps during the reaction.18

Data Analysis of Output Analysis of how to understand cycle threshold is explained inthe Polymerase Chain Reaction protocol online. This will onlyexplain how to read what the software gives. When the run has been completed, the picture below will bedisplayed. There are many features of this software that wedo not use on a daily basis, so we will cover only the basics.See the instruction manual for more information.19

Data Analysis of Output There are many places to start the analysis of your data, butwe will start with examining the Melt Curve. It is critical to use Melting Curveanalysis to determine that youare only amplifying one product.You can also run your sample ona agarose gel and have yoursample sequenced to insure youare amplifying the correctproduct. Sybr green binds to all dsDNA and a melting curve allows youto see when that piece of dsDNA dissociates, including smallpieces of possible primer dimers. By examining the theMelting Curve and seeing that there are only slopes for asmany primers used, you can reassure yourself you are onlyanalyzing the correct product. This is also shown in the Meltpeaks, showing that for the two different primers used, thereare only two different melting peak temperatures.GAPDHMy gene ofinterest20

Data Analysis of Output A print out of all of the data thatwas generated can be obtained byclicking on the “Report” button.The file displayed below is a PDFwhich can also be save and usedon a computer that does not havethe Bio‐Rad software. In this report, the cycle threshold values are display for eachof the wells. From here you can calculate the folddifferences between the genes.21

Fold Change Calculations Before you calculate the fold change of your samples, youneed to examine the ct values generated betweenduplicates. If the values have a large difference ( .5 ct orgreater), you might not want to use them. Also, insure thatyour negative controls do not have a viable ct value. Thismeans that as long as your negative controls have a ct that isdoes not make sense or is a “N/A”, you are OK. The samegoes for the water sample. Fold change is calculated by first averaging the ct values forall samples. Following this, find the delta ct by taking thegene of interest average minus the GAPDH average. Do thisfor treated and not treated samples. After this, find thedelta‐delta ct by taking the treated delta ct value minus thenon treated sample delta ct value. Determine the fold bytaking the delta‐delta ct value (x) and calculating 2 ‐x.22

Bio‐Rad 96‐well plates Bio‐Rad 96‐well plate clear covers EvaFastMaster‐Mix with low ROX is located in two places. The currently used Master‐Mix is located in walk‐in fridge in the “Molecular Biology” drawer while the stock tubes are located in the ‐20 freezer located in the lab. Bio‐Rad Master‐Mix .