We have performed gradient PCR for our primers (Tm:59) with temperatures ranging from 55 to 600C. In Thermocycler, we have observed a graph indicating different temperatures ranging 55-600C (i.e.,55.0,55.1..and so on) along 12 columns. We have inserted 3 tubes at 3 different temperatures( 55.0,57.2,59.9 ) and the time taken was 1hr:42min. But according to few sources, gradient PCR will take around 3-6 hours. Can we conclude amplification was done at those 3 temperatures?
Dear Kamil,
I have done it before many times. This process is called PCR optimization. if you have time and energy, there are four steps to follow:
1. Since your Tm is 59, setting +/- 5 is the best option: i.e such as 52 53 54 55 56 57 58 59 60 61 62 63. And keep Denaturing and Extension constant.
2. Once you found best band at Tm=x where x is 52 53 54 55 56 57 58 59 60 61 62 63, try to optimize denaturing by keeping Tm, extension, and # of cycles constant
3. Then, keep Denaturing and Tm=x constant, use variable extension time (the fastest and best band observed time). Therefore, you optimized your PCR protocol.
4. Using best Denaturing, Tm, and Extension temperature, optimize the # of cycles i.e set cycles 25 27 29 30 33 36
Wish you Good Luck
Teddy
Hi, and you get a specific band at one tested temperature? I don't understand your problem, I guess... Cheers, Nadine
Dear Kamil,
I have done it before many times. This process is called PCR optimization. if you have time and energy, there are four steps to follow:
1. Since your Tm is 59, setting +/- 5 is the best option: i.e such as 52 53 54 55 56 57 58 59 60 61 62 63. And keep Denaturing and Extension constant.
2. Once you found best band at Tm=x where x is 52 53 54 55 56 57 58 59 60 61 62 63, try to optimize denaturing by keeping Tm, extension, and # of cycles constant
3. Then, keep Denaturing and Tm=x constant, use variable extension time (the fastest and best band observed time). Therefore, you optimized your PCR protocol.
4. Using best Denaturing, Tm, and Extension temperature, optimize the # of cycles i.e set cycles 25 27 29 30 33 36
Wish you Good Luck
Teddy
I don't agree with Tewodros. Normally you don't try annealing temperatures in one °C-steps for the first round. That's time-consuming and expensive. Try differences from 3 °C steps. After seeing a band you can play in 1°C steps to optimize your annealing temperature. Cheers, Nadine
I agree with your disagreement with Nadine. But this what I had done and produced clear bands.After I optimized the PCR protocol, I tested 20 species with 200 SSR primers.
Why one °C-steps? I found bands at lower Tm's or middle Tm's or higher Tm's. Therefore, testing one sample across 12 PCR columns at one °C-steps will give you chance to see all the options where you can find clear bands. I let the decision to the question in hand. This is optimization but not decision at first time. These are the two approach:
3 6 9 12 Nadine approach
1 2 3 4 5 6 7 8 9 10 11 12 Teddy approach
In both cases, you run the PCR one time, the probability of missing a clear band with 3 °C-steps will be higher (approx 67%)
Good luck
Believe me Tewodros: I am doing PCR since years... I never heard that somebody try your time-consuming method.
If I were to optimize a PCR protocol I would perform the gradient PCR as described by Tewodros the difference between setting up 4 vs. 12 identical PCR reactions is minimal.
Try to optimize the design of your primers if you designed them yourself.
The annealing temperature and the extension time depends on the polymerase enzyme you are using. If you are using a polymerase enzyme like Taq polymerase which takes an extension time of about 1min/kb your PCR will take more time to finish. On the other hand if you use Phusion High Fidelity polymerase which needs a extension time of about 15-30sec/kb your PCR will automatically finish faster. The gradient PCR will only help you to standardise the annealing temperature that gives you your gene of interest specifically. There is no relation between gradient PCR with PCR time as far as I know. Now coming to your last question, in order to conclude whether amplification has taken place at any of the annealing temperatures, you have to run a agarose gel of all the three PCR products along with a DNA Ladder and see which annealing temperature gives you your gene of interest specifically. Hope this helps.....best of luck with your experiments. Cheers.
You can please download pdfs of the files attached herewith for getting valuable informations
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