Long RT for 4.5 kb cDNA - (Feb/21/2006 )
Hi,
I want to make a full length cDNA around 4.5kb from RT-PCR, tried several times, I could not get the band. I heard can make up to 10kb cDNA, but I don't know what went wrong.
Does anyone has experience to make a long RT-PCR for this size? Could share your experience?
I use standard protocol RT protocol, superscript II 42C for 50min, use oligo dT for primer.
Many thanks for helps
Che
How about checking the quality of your RNA? Is your enzyme still working (positive control reactions possible for both these issues)? Have you added Rnase-inhibitor to your reaction?
The husbannd of a colleague has gone up to 9,6 kb with transcriptor from Roche (but in my hands this enzyme hasn't worked as good as superscript III). I regularly go up to 3,8 kb with Superscript III, and colleagues have done the same with Expand RT from Roche.
The husbannd of a colleague has gone up to 9,6 kb with transcriptor from Roche (but in my hands this enzyme hasn't worked as good as superscript III). I regularly go up to 3,8 kb with Superscript III, and colleagues have done the same with Expand RT from Roche.
Many Thanks for reply.
Yes, I added RNase Inhibitor, RNA was prepared by Trizol, and DNase treated before the RT. Positive control seems working but only 500bp.
According to your experience, should I need to make 2hr instead of 50min for RT reaction. I only got superscript II at the moment, just wonder how you made your 3.8 kb worked, any more tips I could follow?
Your kind help will be appreciated.
Che
I go up to 3,8 kb with a 30 minute incubation @ 55°C with superscript III, 50 minutes should be enough.
Is the subsequent pcr optimised and sensitive? Maybe this is your problem?
Is the subsequent pcr optimised and sensitive? Maybe this is your problem?
You are probably right, at the moment, I used the Promega Taq DNA polymerase (M1861), but now, I start to worry this enzyme may not be able to make DNA for more than 4 kb from cDNA (although it worked for under 1kb in my hands and I believed it should work for the size of 4.5kb).
What enzyme you used for your PCR ( Taq or VENT, pfu)?
I am going to try all of them to see there is any difference.
Thank YOU so much for sharing!
I've used Platinum Taq high fidelity from invitrogen, pfuUltra from Stratagene, pfx from invitrogen, people here have used expand high fidelity from Roche for years...
I wouldn't bother to try them all, just order one polymerase that (according to the manufacturer) is capable of longer transcripts.
But: if possible, optimise your PCR first... Make sure that you know the detection limit of the PCR reaction itself, and when doing the actual RT-PCR, include a positive (DNA) control, then you can deduce if the problem is your RT or your PCR.