kit - https://www.qiagen.com/us/shop/sample-technologies/rna/rneasy-powerbiofilm-kit/#orderinginformation

qPCR protocol - Article qRT-PCR of microbial biofilms

Thank you Abhijeet. I will try to get hold of this kit.

I treated the m. tb cells with lysozyme for 2 hours at 37 degrees followed by qiagen kit for RNa isolation .

Hi Manoj!

Did that work for you? how much was the starting cultures and the final yield.

I used to take pellet from 5 ml of culture (H37Ra). After centrifugation @3000 g for 10 minutes, i treated the cells with 1.5 ml of protoblstic buffer containing 4 mg/mL of lysozyme. after incubation at 37 degrees for 1 hour, the pelleted protoblasts were then subjected to processing by RNeasy Kit from Qiagen. in my case, The final yield of RNA varied from 323 - 832 ng/ul for the samples processed together.

The important step to increase the yield is that after adding 1 mL of RLT buffer and b-mercaptoethanol to the suspension , use glass beads 25-50 mg for lysis @ 3000 rpm for 5 minutes. Thanks. Gudluc

Thank you for the information. I am trying different things based on the availability of reagents. I can also give this a try.

Hi!

One more thing, can you share the composition of your protoplast buffer. I use 10mg/ml lysozyme in TE buffer for isolation of RNA from M.marinum and keep it on 37 shaking for 2hrs . I get

~1ug of yield from 20ml of log cultures. I am using trizol method for this purpose. The yield is not as good but purity is fine to work with.

15mM trisCL pH=8, 0.45 M sucrose, 8mM EDTA, 4 mg/ml lysozyme : composition of protobladtic buffer. I

I didnot use trizol. i just use the Qiagen kit.