LR reaction - Cloning into a expression vector (Dec/05/2009 )
Hi, my name is Jose and I am trying to transform barley using RNAi. I am having problems with the LR reaction. I am using the invitrogen gateway protocol, but so far I can not identify my gene into the expression vector. I do not if following step by step the protocol is good idea and I am sure that there are many modification working better. Could somebody please let me know if they are experimenting problem???
I am trying to identify my inserts using a vector specific primer and my gene specific primers and I am getting good products, however, the same band is observed in samples where insert is not present, therefore it seems like I am aplifying something else. In addition, I digested and the band pattern is no the expected, as the enzyme just cut once when at least 5 were expected. There is any problem when performing this sort of cloning??? Is it the plasmid affected by any kind of modification???
Thank you
tcimisces on Dec 5 2009, 02:56 AM said:
I am trying to identify my inserts using a vector specific primer and my gene specific primers and I am getting good products, however, the same band is observed in samples where insert is not present, therefore it seems like I am aplifying something else. In addition, I digested and the band pattern is no the expected, as the enzyme just cut once when at least 5 were expected. There is any problem when performing this sort of cloning??? Is it the plasmid affected by any kind of modification???
Thank you
Generally LR reactions are fairly easy and you shouldn't have any trouble identifying the right clones if you get lots of colonies. However, someone in our lab was doing a microRNA experiment using the LR system and he repeatedly was unable to get what he needed. Several people tried it as well and it always came out goofy. In that case, I believe that my labmate had to redesign his primers to make the donor clone.
The PCR you describe sounds like it's just giving you false positives - something that happens frequently. I would probably try doing a restriction digest with some other enzyme to make certain that the clones really are incorrect and maybe, if you can, sequence one of the clones to see what is going on. Of course you should also double check your plates, make sure you're using the right antibiotic, etc. Once you exhaust all of these possibilities, then you might want to redesign your donor clone.