I am trying to clone HSP90.1 cDNA. For that, I have extracted RNA of Arabidopsis from the leaf. From RNA, cDNA is formed and used for PCR using primer. But I am not getting anything. I have tried different dilutions of cDNA and even tried different polymerases but neither get any specific or non-specific band. cDNA is formed correctly as I have checked PCR of 26s rRNA and I got a band.
One of my friends told me that there is the possibility that hs90 does not express in leaf. Can anyone tell me from which part or in which condition of arabidopsis should I use to get high level of hsp90 cDNA.
Hii, I hope you are doing well. Could you explain a little bit how did you design primers ? Sometimes to copy total gene sequence, you should use adaptor sequences that gives stable binding ability to primers . Maybe primer binding is insufficient or pcr optimization must be applied.
Hello. You can design primers on the web interface of Primer3 tool or you can use Primer1 tool too for TETRA-ARMS PCR. In both of the interface, you can create optimized primers of your choice by tailoring the dependent or independent factors.
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