Positiv "Negativ control" using NEXTflex Small RNA Kit

[CarloB]

Hi,

I hope someone can answer my questions and bring some light into my darkness.

I am using the NEXTflex Small RNA Kit in CLIP experiments to analyze mRNA binding motifs. So I immunoprecipitate RNP, do a proteinase K treatment, and precipitate previously bound RNA by TRI reagent. It work well so far and after using that Kit I got amplified products between 140 and 180 nts.

So far, so good.

Yesterday I though I might need a positiv and a negative control, respectively ... just checking! So I used the microRNA control (part of the kit) as positive control and added just water as negativ control (for ligations steps). After the second ligation I gel-purified my products by using a 12% UreaPA gel ... mainly to get rid of the adapterdimer. Unfortunately, I obtain a "postive band" in both samples after PCR, means for the 21 nt microRNA and the negative control "water".

How can that be!? My problem is that I already got some products for sequencing, but now I am not sure if I have real data or just this "postive negative control" thing. Is someone able to advice me somehow!? That would be endless generous

Thanks a lot.
Best, Carlo

[Bioo Scientific]

Hi Carlo,

The positive control miRNA should give you a band size of approximately 146nt. The adapter dimer should be 120 nt. If you email me your positive and negative control gel images to: [email protected] we should be able to help you.