I am trying to confirm my transgenic plants by using RNA dot blot. Can anyone tell me that how can i denature my RNA samples, which concentration of RNA should i use? and how can i make dilutions with denaturing buffer.
Hi Imran,
try to prepare your total RNA with a final concentration of 10 mg/mL, then use up to 5 µg per dot. Before loading on the membrane (we use Hybond N), denature the RNA in a formamide/formaldehyde/MOPS mixture at 65°C for 5 min, then cool on ice and put in your membrane buffer. Dry membrane after application and continue with probe hybridisation as with Northern Blot.
Hope this was helpful! Jessica
What transgene are you testing? Some of them you can directly test on the gene products (proteins) with a commercial strips. Ex. nptII (kanamycin-resistant gene) or Bt gene ImmunoStrip.
https://orders.agdia.com/immunostrip-for-btcry1ab-1ac-stx-06200
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