Amplifying a 6.5 kb cDNA - (Aug/30/2006 )
I am working with Arabidopsis, and I would like to amplify a 6.5 kb cDNA to clone it and complement my mutant to show that indeed the mutated gene is responsible for the phenotype of the mutant.
I have extracted RNA using a standard 2 days protocol using LiCl. Then I treat the RNA with DNase (RQ1 RNase-Free Dnase, Promega) and do RT-PCR (M-MLV Reverse Transcriptase, promega) using 5µg of total RNA and random primers for 1h at 42°. The cDNA are OK since they work for a regular PCR amplifying a 400 kb fragment. Nevertheless when I try to amplify my cDNA using a ABgene Extensor Hi-Fidelity PCR Enzyme (supposed to amplify uo to 20 kb) I get nothing (except for one time where I amplified a weak band that I could never obtain again...
What would you suggest to me to try to amplify and clone this cDNA ??
Thanks in advance .
If your cdna conversion is ok, it must be your pcr and specifically, you should look at your primers. Is the annealing temp too high? is your extenstion time at least 1.5min/kb (long enough for a 6.5 kb fragment)? Verify the correct primer sequence.
If you use random primers, shouldn't you have a lot of short cDNAs? Are you sure that with these primers you can get a template that is long enough for your primers to bind and to get the full length product you expect? I would prefer the oligo dT for the RT
Thanks for your answer, I am using a polymerisation time of 15 min so I guess it is sufficient. Concerning the RT I thought that opposite to what you said when you want to amplify a large fragment you should better use random primers because with oligodT you have fewer chances to amplify the whole fragment ??
Otherwise I thought of extending the RT time to 2h or even 3h, or may be starting directly from my gene specific primers and not using the random. Do you have any experience in that kind of experiment ?