HELP! cloning cDNA from double-stranded cDNA synthesis - (Mar/14/2006 )
Hi,
I am entirely frustrated and thought I'd appeal to this community for help-
I have amplified mRNA from a prokaryotic community and have microgram amounts of decent mRNA. I then used Invitrogen's double-stranded cDNA synthesis kit which ends in spiking the reaction with T4 polymerase so as to render the cDNA ends blunt. I am having absolutely no luck getting any clones when trying to clone into a blunt vector. All the cloning controls work as expected and the transformation efficiency of the comp. cells is fine, but I can't get any clones with my cDNA. I'm starting to wonder if there's a problem with the cDNA quality, but how can I assess quality? It looks like a shmear on a gel (which is expected since it's all sizes, up to ~1.5 Kb) and the concentration is reasonable. Why won't it clone??? Suggestions?
Thanks,
Rachel
I am entirely frustrated and thought I'd appeal to this community for help-
I have amplified mRNA from a prokaryotic community and have microgram amounts of decent mRNA. I then used Invitrogen's double-stranded cDNA synthesis kit which ends in spiking the reaction with T4 polymerase so as to render the cDNA ends blunt. I am having absolutely no luck getting any clones when trying to clone into a blunt vector. All the cloning controls work as expected and the transformation efficiency of the comp. cells is fine, but I can't get any clones with my cDNA. I'm starting to wonder if there's a problem with the cDNA quality, but how can I assess quality? It looks like a shmear on a gel (which is expected since it's all sizes, up to ~1.5 Kb) and the concentration is reasonable. Why won't it clone??? Suggestions?
Thanks,
Rachel
Hi Rachel,
Your transformation is obviously okay, and presumably you have included a blunt-ligation control (i.e. religation of SmaI-cut vector) to confirm the ligation is working? You must have used randomer priming for both the RT and second-strand synthesis; did your protocol include E.coli DNA ligase to close the nicks left at the 5'-end of each randomer in the second strand?
Most likely problem:
If your polymerases (Klenow for 2nd strand synthesis or T4 polymerase for polishing ends) aren't fully inactivated after completing the 2nd strand synthesis / blunting, and especially if the dNTP concentration is low (very likely if you started with lots of RNA) they will readily remove bases (past blunt) from 3' ends. This will leave 5'-overhangs, which won't clone... To resolve this, use less enzyme, add additional dNTPs after second-strand synthesis, keep the temperature low during blunting (12oC max. for T4 polymerase), add extra EDTA (10mM final conc.) to the reaction before heat-inactivation of the polymerase/s, and try ligating immediately after blunting. It might also be worth repeating the blunting with mung bean nuclease (removes 5'-overhangs) just to make sure you have ligatable ends.
Hopefully this will fix the problem.
Another possibility:
Assuming you're working with prokaryotic total RNA, >95% of your RNA will be rRNA, with lots of secondary structure. I'm not sure, but I suspect this could affect synthesis of full-length second-strand cDNA from your target mRNA. You could try removing the excess rRNA first by probe-capture. Ambion sell a kit for this:
http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=246
This won't necessarily remove all of the rRNA from unknown members of your community, but it should remove quite a bit, and increase your chances...
hope that helps, and good luck!
Del.
Thanks, Del! I agree that the problem is with the ends of my cDNA. It seems difficult to guarantee that both ends are blunt (or not).