Hi all,
Currently, I am having difficulty with Gateway LR cloning. Actually with only one gene. For other genes, the technology works almost %100. However, when I tried to transfer HDAC2 into my destination vector (pFRT-TO-DEST), cloning fails and I don't get any colony. However, other genes I tried to clone at the same time worked well. Have you had similar problem with LR cloning? Where would it go wrong?
P.S. I use Invitrogen LR CLonase II. I follow the exactly same protocol written in the instruction. I also tried to extend reaction time.
Cheers,
Hi Mert,
I have a few suggestions, recheck your entry clone for concentration and purity there may be inhibitors that impede the LR clonase or miscalculation in the entry:destination ratio. You can also try to linearise your entry vector by using a restriction enzyme somewhere in the backbone away from the HDAC2 and recombination sites. After the digest and purification try the LR reaction again.
All the best
Hi Joshua,
Thanks for your answer.
1- I have checked concentration of both entry and destination vectors. They are correct.
2- In my multiple trials, I tried to use both recommended amount of plasmids and equal copy number.
3- Besides, I sent the entry vector to sequencing to ensure no any mistake in the sequence, especially in the attL sites.
Next I can try to linearise the entry vector and try again as you suggested.
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