Has any one successfully performed miRNA cloning? - miRNA cloning troubleshooting (May/14/2007 )
I am trying to clone miRNAs. I am using the protocol in "molecular biology protocols". I tried it 3 times and failed 
It is so frustrating that after going through 10 days of experiment you dont see anything. Is any one out there who is doing miRNA cloning and wants to share his/her experiences? Thanks in advance.
Which step of the protocol didn't work?
Andrea,
thanks for the reply. For me, ligation of 5' and 3' adapters seems to be working. But i am getting only very faint bands of rt-pcr products. Also there are three bands instead of one and one of them has exactly same size as i was expecting. No bands in the negative control. They could be real bands, but what concenrns me is why so faint bands? i ran the pcr for 40 cycles.
94 - 2 min
94-15 sec
50- 60sec
72-20 sec
72-10 min.
I am using platinum taq invitrogen. i think its the pcr problem. should i go for lower annealing temps? or increase time?
thanks in advance.
If you run an aliquot of your pcr, a few microliters out of a 100-200 microliters reaction, the product may be faint, even barely visible when stained with ethidium bromide, and yet give a good library.
There should be a single band, sligtly smeared. Different bands may be amplified artefacts coming from your pcr, which is too long (I wouldn't do more than 25-30 cycles, and wouldn't lower the annealing temp), or, more likely, from the upstream adapter ligations and gel fractionations. In case of multiple bands, it would be better to gel purify the correct one, although this will involve some loss of product. You could try to re-amplify the purified product, but I have no experience about it, and it doesen't sound nice to me because I think the amplification should be kept to a minimum. Before the pcr, it is important to size-select the adapter ligation products in a precise way and purify them well, avoiding any possible inhibitor.
Hope this may help, good bye
Andrea
There should be a single band, sligtly smeared. Different bands may be amplified artefacts coming from your pcr, which is too long (I wouldn't do more than 25-30 cycles, and wouldn't lower the annealing temp), or, more likely, from the upstream adapter ligations and gel fractionations. In case of multiple bands, it would be better to gel purify the correct one, although this will involve some loss of product. You could try to re-amplify the purified product, but I have no experience about it, and it doesen't sound nice to me because I think the amplification should be kept to a minimum. Before the pcr, it is important to size-select the adapter ligation products in a precise way and purify them well, avoiding any possible inhibitor.
Hope this may help, good bye
Andrea
Thanks a million andrea! I reduced the number of cycles to 30 and i am getting only one specific band. I want to clone it. Do you have any experience in cloning such a short (~60 bp) PCR products? How much of the total pcr product should i use for ligation in to a TOPO vector for example? Could you or any one give me some tips? I dont want to gel purify the product as the band is very faint and i might lose most of it.
thanks a bunch.
Hi Polsum,
Have you get your RT-PCR work well?? I get 3 bands like what you said. But i got very intensive band of around 35-40bp. I keep trying adding DMSO, increase the annealing temp to 60, order new primer from different company, but i still cant get the single faint band at 50-60bp. My pcr cycle is 30 too.
Hope to hear you soon.
Thanks.