Could you try a longer drying spin, i.e. 3 min rather than 1 min, at step 5?

Could you try a longer drying spin, i.e. 3 min rather than 1 min, at step 5?

Hi Simon. Thank you for your suggestion, which is a really sensible one. I'm thinking of letting it air-dry at step 5 for around 10m. May be I'll try both and see how it goes.

No problem Thanh

I have always liked the air drying spin step as its a very 'active' process.

Good luck, Simon

Sometime we just open put tube in incubator for 10 minutes (40ºC)

let the spin column to air dry for 5 min. also let the RNase free water for 2-3 min in the spin column before gentrification  

Hi,

I usually pipette out the ethanol without touching the matrix before the dry spin for 2 or 3 min.

-Ram

Hi Thanh

You can also consider to skip DNase step since the RNAeasy kit is optimized to avoid content of gDNA contaminants in your final sample.

In this case, if you will perform any PCR using cDNA templates synthesized from RNA, just remember to perform parallel amplification using a gDNA sample as a control. If the gDNA control amplification will result in bands at higher sizes together with your amplicon band or if no bands will appear when compared with the cDNA amplification, this will confirm lack of significant gDNA contaminants in your cDNA samples.

Otherwise, if you decide to perform DNase steps, I recommend Simon's suggestions above.

Good luck

AMC

1-Spin longer with rhe led open for 3 min At high speed.

2- Be carful when discard the collection tube, do not make the column toach the collection tube.

It's a common problem since Qiagen changed the protocol to say that you don't need to change the elution tubes between the washes.

In my personal experience, you can get ethanol and also salt from RW1 sticking around the outside of the column, so I change the elution tubes (you can get them separately) and also dip the columns on a paper towel to remove liquid on the outside of the tube.

And in particular, use a fresh tube to do the dry membrane spin!

Hi everyone

Thank you very much for your insights, it is definitely of a great help.

I changed new tubes after step 3 and 4. And after spinning at step 5, 12000g 5m, lid open; samples are confirmed to get rid of EtOH and successfully loaded on gel.

Bravo!