Hello! My primers dont attach to their true place in paraffin-embeded tissue samples and make dimmer primer with each other. How can I fix this problem so my primers connect to their gene? Please answer me soon. its very very very essential for me. Thank you a lot
There are multiple things you can try.
Did you for example try out a higher annealing temperature? Do you have a non paraffin-embeded sample on which you can try out your optimization attempts (proper positive control)? Sometimes the coice of PCR mastermix also influences the performance and trying out a different mastermix or adding PCR enhancers (e.g. DMSO) can sometimes help.
In worst case you need to re-design your primers.
Anna Schötta
Thank you very much for your recommendations. Yes I tried my primers on fresh tissue samples, my primers attached to their gene and I had true melting curves. But in paraffin-embeded tissues I just have dimmer-primers. sorry can i ask one question about DMSO? the final volume of each microtubes solution (mastermix+ DW+ primers+ tissue sample) is 20 microlittr. How much DMSO do you prefer for this volume?
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