2 replies to this topic

[chimmi]

Hi friends....

iam in bit of hazzle now ....as per my guides suggestion i used the genomic DNA amplified gene to be cloned in pGEM-T EAsy vector and then used the same for constructing dsRNA by invitro transcription using T7 and sp6 RNA polymerases. now the gene size i have used for invitro transcription is ~801bp and 895bp. but when i run the dsRNA product after annealing the two ssRNA conctruted separately by T7 and Sp6 polymerases using T7 and SP6 primers as they are vector primers. the product of the dsRNA i get in agarose gel is ~80bp. How can this happen??? If transcription has been done, the size of dsRNA shud be of same size as that of the gene size???? Am I right or does this not happen   when we do invitro transcription???? will the size of dsRNA vary??? got a presentation and iam going to get fired up with the questions when i show the gel picture.please help.   please check my gel and reply me.... the marker run is dsRNA starts with 21bp,30bp,80bp,150 bp,300 and 500bp. 21 and 30bp not to be seen.

Thanking you

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Edited by chimmi, 22 May 2009 - 03:09 AM.

[bob1]

Primer dimers, you haven't actually produced your gene.

[chimmi]

bob1, on May 25 2009, 05:53 AM, said:

Primer dimers, you haven't actually produced your gene.

Primer dimers are degraded when treated with DNase i suppose. Coz the whole mixture is treated with DNase after annealing step.....so can that be primer dimers????