I am doing Multiplex PCR, which is already standardized at annealing temperature 55 C. I am running mice DNA in PCR.
I got good amplification of one sample which is control (Original DNA concentration = 32ng/ul; Template DNA concentration = 160 ng/ul) .
While I performed the similar PCR with other samples :
1. Original DNA concentration = 46.3 ng/ul; Template concentration = 231.5 ng/ul
2. Original DNA concentration = 28.7 ng/ul; Template concentration = 143.5 ng/ul
3. Original DNA concentration = 43.7 ng/ul; Template concentration = 218.5 ng/ul
4. Original DNA concentration = 58.3 ng/ul; Template concentration = 291.5 ng/ul
5. Original DNA concentration = 69.7 ng/ul; Template concentration = 348.5 ng/ul
6. Original DNA concentration = 53.5 ng/ul; Template concentration = 276.5 ng/ul
PCR conditions are:
95 C - 5minutes
95C-0:30 sec
55 C-0:30 sec
72 C-0:45 sec
72 C-10 minutes
But did not get any amplification in any of the samples except control.
Please suggest where i am lacking in order to get my desired bands.
Hi,
multiplex PCR can be tricky. I am not sure from your description if you expect different bands in the samples (I guess due to multiplex PCR). Did you already check if the different primer pairs work at this conditions? You could try it once again but this time just use 2 primers per PCR. Somtimes primer pairs work but multiplexing does not. Another idea is to run a gradient to find the optimal annealing temperatures for the respective primer pairs. You can also play around with the MgCl2 concentration (the higher MgCl2 the lower the specificity). Another option is to try different DNA concentration. But only change one parameter at a time that you know in the end the effect of the respective parameter.
Good luck!
Dear Thomas Svoboda. Thank you for the suggestions.
I have run the gradient PCR also (Picture attached). as the Tm of the three primers is
1. 59.0
2. 59.7
3. 59.1
So I provided the temperature range from 57 C to 60 C. But I found the one band start disappearing of emrgeing with other band. That is why the temperature is 55 C.
1. I will surely do PCR with different template concentrations first condition.
2. I will try PCR with two tubes with second time , if it requires.
If any suggestion, please let me know. As I am doing the PCR with first condition today. If you can suggest me the template concentrations as I have given the original concentration also. that will help me two decide three or four different template concentrations for PCR@
Thomas Svoboda . I am expecting only either of these two bands in all the samples, which showed in the first picture of the gel in control (two bands). That is why i used this sample as control.
Thanks
As far as I can see from your gradient gel at 57°C you only have one band left but maybe you can also go lower. As a rule of thumb you can assume that the annealing temperaure is tm - 5°C. Concerning the DNA concentrations I would suggest to try to insert different amounts between 50-200 ng in a 50 µl PCR reaction. If multiplexing does not work properly do the PCRs with 2 primers respectively. This way you should clearly see the difference between the control and your candidates (if the previous modification worked).
Thomas Svoboda Sir, thank you very much for the advice. I performed the template concentration standardization and find out the best annealing temperature. I got my PCR standardized. You suggestions helped a lot.
Thank you!
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