4 replies to this topic

[helene]

Hi everybody!

Since several months, I perform a RTPCR using 12 µg of total RNA as template for RT. For the PCR I used 2µl of the RTproduct. The RTPCR worked very well and gave 5 bands .3 of them were cloned. surprisingly, the 2 others could'nt been cloned.

Since one week, I use 1µg mRNA (isolated with the kitmicrofast track form invitrogen)as template for the RT and I use similar  quantities  of cDNA as previously but  I obtain poor amounts of PCR products  and the bands that I couldn't clone before disapear.

Does  someone have a solution or explication for this problem? ? Thank you very much

??helene

[milanoj]

The extra bands you are seeing may be an artifact of gDNA contamination. No RNA isolation method is perfect. When you isolate mRNA the contamination disappears. Were you expecting all these bands? I would isolate the extra bands then reamplify them to get enough for cloning. Or sequence them without cloning.

[helene]

 Hi Milanoj!

I've already tried isolating the bands of the gel and reamplify them .But when I do this, I obtain all the five bands as if I've  not isolated the bands of interest. The negative controls of RT (PCR with RNA and RTPCR without reverse transcriptase) are negative...so I think that there are'nt genomic contamination!

  helene

[scogne]

Hi,

I think the 2 bands you got are due to one oligo amplification only. Did you try to check them separately on the total RNA cDNA?

[Sambucus]

Have you tried a DNAse treatment? You could specifically eliminate any contaminating genomic DNA before the reverse transcription.

The occurance of DNA in a total RNA isolate usually means you are using too many cells. Try using less tissue, or more extracting media.

Increasing the annealing temperature in the PCR reaction can dramatically reduce any artefacts from the amplification, due to mispriming.