Dear all,

Hope you were fine there in this Pandemic situation. I am trying to amplify the acdS gene (ACC deaminase encoding gene) using bacterial gDNA as a template. But it did not amplify and even on agarose gel it does not show the Template on visualization, I did not understand the reason behind non-amplification of my gene and why my template did not show after amplification which needs to be visualized on Gel dock. Kindly help me to overcome the issue or help me by giving the cycle which you used for amplification. I used Geneaid Taq polymerase, Thermo (2X) Master Mix, and Takara (Emarald Amp- Mastermix). The primer which I am using is (F)- 5’GGCAAGGTCGACATCTATGC-3’ (R) 5- GGCTTGCCATTCAGCTATG-3 (amplicon size around 1kb) The Programme which I am using is for Taq Polymerase Initial Denaturation- 94 C- 4 min 35 cycles of Denaturation- 94 C- 30 sec Annealing- 48-59 C- 30 sec Extension- 72 C- 45 sec Final Ext- 72 C- 10 min

and for Mastermix (Thermo & Takara) as per the manufacture instruction with slight modification for Ta determination. Note:- I used 10picomole/microlitre primer concentration.  I also had another set of primer as-

acdS (Universal) Primer set

F(5′-GCCAARCGBGAVGACTGCAA-3′

R (5′-TGCATSGAYTTGCCYTC-3′

having amplicon size around 800bp.