Amongs all these responses I have a question to make: someone suggested oligo dt someone else random primer. I have a 4000bp cDNA whith wich I am having problems to amplify with specific primers that I designed bringing my restriction site sequence. What have I use to get the amplification product???
Cloning greater than 3000bp cDNA will be a challenge independent of whether u use oligo dT or random primers, especially if the target mRNA is bad expressed. You will have to do many things right to get it to work. A RNA source with good expression and a better Reverse transcriptase suited for longer transcripts (I know some from Invitrogen) will be most important and once you have it you can try your luck with both oligo dT and random primers.
You can also try amplifying and cloning pieces separately and join them later.
Hi Manoj, amplifying and cloning pieces separately you mean that, starting from the cDNA, I have to amplify piece of 400-500bp with specific primer, until I will cover all the length of my cDNA, and then make a ligation between them???
yes, i mean random primed cdna and trying to clone less than 1500bp fragments. Not ligating fragments, but subcloning after u get them cloned separately. Only If the full length pcr is not working. Good luck.
Manoy sorry but when you say random primers you mean that I have to design specific primers ( two pairs for example) for my cDNA. For example one pair may be that one I am using bringing my sequence for restriction enzyme and another pair can fit in the middle of the cDNA sequence, one 1000 downstream the 5' and the other one 1000bp upstream the 3'. Does i sound good? be more clear as I am not properly an expert.
I mean random-primer for reverse transcription (to get at least partial cDNA fragments from your large mRNA). Obviously for cloning you will have to do PCR with gene-specific primer pairs (may be 3x separate pairs for amplifying portions of your 4000bp separately). Hope it helps.
Why isn't it possible to do a methylation of the restriction enzymesites in the desired cDNA when you use specific primers for a desired gene in that cDNA?
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