Hi Atul,

I am not convinced that you have a problem with DNA contamination because you cloned a transcript which retained only 1 of 11 introns. Thus I would suggest that this alternatively spliced transcript is expressed in your sample and may represent the major fraction. Have you seen several DNA bands in the agarose gel after PCR? You can try to sequence more clones as suggested by Ziguo Zhang but I would also try to amplify the transcript from another sample. Nevertheless you should remember that the alternative transcript may represent the "important" transcript.

Good luck

Dear Atul

you must have amplified something from your genomic DNA contaminating your extracted RNA and thence cDNA

• To prevent amplification from DNA you need to situate primers across exon exon junctions
• The most effective way to do that is to identify your various gene transcripts in Gen Bank and utilise the associated primer design tool to situate primers across an exon exon junction
• A method for identifying your transcript of interest is provided in the attached SOP
• Also, if at all possible situate your primers with 2kb of your Transcript terminus (poly A tail) to ensure efficient production of cDNA
• In addition, having designed the primer across an exon exon boundary BLAST the primer to ensure it will only bind to your RNA transcript (in cDNA) and not genomic regions with introns:
• https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
• All of these procedures are described in the attached SOP

I used to routinely digest my RNA with DNase I as I found that this was the only way to definitely get rid of genomic DNA contamination after RNA extraction.

screen more colonies, or try to get rid of the intron by plasmid modification method.

Hi Atul,

I am not convinced that you have a problem with DNA contamination because you cloned a transcript which retained only 1 of 11 introns. Thus I would suggest that this alternatively spliced transcript is expressed in your sample and may represent the major fraction. Have you seen several DNA bands in the agarose gel after PCR? You can try to sequence more clones as suggested by Ziguo Zhang but I would also try to amplify the transcript from another sample. Nevertheless you should remember that the alternative transcript may represent the "important" transcript.

Good luck

Good point Norman. In my experience too transcripts are tissue specific so perhaps as you say they are finding what they are meant to find

Atul: Assuming this is a veritable result then you might want to saturate the sequence the whole thing and clarify its exact nature. Still not sure about the inclusion of an intron though: Cloning and sequencing the whole insert followed by BLASTING would be wise

you may also try to use ensemb.org instead Gene Bank

Agreed Susanna. I look at both and take as my starting point transcripts common to both models 

yes, especially because ensembl has "splice variants" and it's much easier to separate exons and introns

Hey Suzanna. Genbank does too but the models used to derive transcripts. Ensembl uses in house annotation scripts whereas genbank takes data from outside including EST RT PCR data. Thus looking at transcripts common to both is quite a good idea because you are independently confirming transcripts from multiple data sources derived using different methods