Previous when I did the qPCR with Bio-rad iCycler iQ™ Real-Time PCR Detection System, I can get the PCR efficiency between 95%~105%. Recently, our lab introduced the Bio-rad CFX 96. However, when I carried out the qPCR with the same primer, template and SYBR green mix, the PCR efficiency was lower than 90%. I did not know what happened here.
key points to be checked
Integrity of primers and DNA (these two are crucial among all)
Proper storage of SYBR mix and compatibility of mix to machine(product info sheet)
Machine to machine variation in temp. ramp rate
If needed you can standardize your primer pair in new machine.
This wont take long but you will see better result.
You have two different machines now. In which you are facing problem from new one.
The same primer reaction with set of replications (possibly most identical reactions) can be kept in both machines simultaneously. see if any observable change between both. If it is so you need to check for compatibility of SYBR mix to your new machine.
Hi jinzhi, in m experiment.. new primer or new machine when use we will do steps follows:
1: confirm the primer temperature in the new machine cause different machine may have different system that you should get attention...
2: cause the new machine the primer never test so is better to check the melt curve even the time will be longer...
may help you...
Do you use the same 96 well plates as before? When we switched to the Cfx96 we changed our plates, too, and had to adapt our programs because the transmission of the temperature was different (perhaps the ramp rate, too, I never checked).
Hi Jinzhi,
Are you using the thermal cycler´s software to calculate PCR efficiency? Maybe the software version is different, so I suggest you check the software version of both thermal cyclers.
You could also do what Lakshmi suggest, run the same reaction on both machines and see if the Ct values that you get are so different.
There are also alternative programs to calculate the efficiency independently of the thermal cycler, like "Real-time PCR Miner". http://ewindup.info/miner/
Hope it helps.
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