Problems in concatamerization and Taq end filling - (Aug/03/2008 )
Dear all,
Hi, i am having problems in concatamerization of microRNA ligated with linkers as refered to the Bartel protocol.
I used NEB T4 DNA ligase to ligate the linker-ligated microRNAs at 23C for 30 min and i get very very faint and sometimes no concatamers on my gel. Do you guys think overnight ligation at 16C is better? Or 30min at 23C is too long or too short?
And, is the Taq end filling effective? i did it at 1x PCR buffer, 4mM mgcl2, 0.2mM dNTPs mix at 72C for 30-60 min, and my cloning totally failed 
..
What do you think??
Taq fill in usually works well. I did it many times with 1x pcr buffer, 2.5 mM dNTPs, 1.5 mM MgCl2, 72 °C for 15 min. Since we only have a hotstart in our lab, I activate the taq before use.
I had to repeat twice the concatamerization. The first time I tried a protocol from Ambros lab (perhaps originally from Tuschl lab) with a pre-incubation step with the pcr primers, but in my hands it didn't work. The second time I followed the Bartel protocol, but with 1 h incubation at room temperature. In the gel the concatamers looked like a smear between 60 and 100bp, another stronger smear between 100 and 300, and no dna was visible above 400. I extracted and cloned a slice between 300 and 1000 bp.
I had to repeat twice the concatamerization. The first time I tried a protocol from Ambros lab (perhaps originally from Tuschl lab) with a pre-incubation step with the pcr primers, but in my hands it didn't work. The second time I followed the Bartel protocol, but with 1 h incubation at room temperature. In the gel the concatamers looked like a smear between 60 and 100bp, another stronger smear between 100 and 300, and no dna was visible above 400. I extracted and cloned a slice between 300 and 1000 bp.
Dear Andrea,
Thx. Is that mean you pre-incubate the Taq at 72C before add in the concatamer?
For concatamerization, do you use low melting agarose to extract the concatamers? I gel purified the BanI digested RT-PCR product on a page gel to remove the small fragments of the linkers so as to prevent re-ligation of the linkers fragments during concatamerization. But gel purification from PAGE seem make me lost a lot of BanI digested cDNA. And i can only get a very very faint smear after concatamerization on a PAGE gel, stained with SYBR gold which is 10X more sensitive than EtBr. Is this mean i lost a lot of cDNA and concatamerization can be done? I really confuse.
The gel purification is soaking the page gel slice in 0.4M NaCl at 4C overnight to elute the BanI digested cDNAs. Is this method effective for cDNA? As i found this method good to gel purified the small RNAs after linker ligations.
Ask a lot. hahahah
Thanx..
I had to repeat twice the concatamerization. The first time I tried a protocol from Ambros lab (perhaps originally from Tuschl lab) with a pre-incubation step with the pcr primers, but in my hands it didn't work. The second time I followed the Bartel protocol, but with 1 h incubation at room temperature. In the gel the concatamers looked like a smear between 60 and 100bp, another stronger smear between 100 and 300, and no dna was visible above 400. I extracted and cloned a slice between 300 and 1000 bp.
Dear Andrea,
Thx. Is that mean you pre-incubate the Taq at 72C before add in the concatamer?
For concatamerization, do you use low melting agarose to extract the concatamers? I gel purified the BanI digested RT-PCR product on a page gel to remove the small fragments of the linkers so as to prevent re-ligation of the linkers fragments during concatamerization. But gel purification from PAGE seem make me lost a lot of BanI digested cDNA. And i can only get a very very faint smear after concatamerization on a PAGE gel, stained with SYBR gold which is 10X more sensitive than EtBr. Is this mean i lost a lot of cDNA and concatamerization can be done? I really confuse.
The gel purification is soaking the page gel slice in 0.4M NaCl at 4C overnight to elute the BanI digested cDNAs. Is this method effective for cDNA? As i found this method good to gel purified the small RNAs after linker ligations.
Ask a lot. hahahah
Thanx..
Hi Yew,
for the fill in I incubate the taq, in pcr buffer 1X, at 95 for 10 min before adding it to the concatamers, but I think you don't need to do it if you have a normal taq (not hot start).
I didn't purify the digestion on a gel. As I tried unsuccesfully a strategy to prevend re-ligation of the digested fragment (the preincubation with the pcr primers; the protocol is on protocol-online), and succeded just going straight to the concatamerization, I tend to think that these strategies are not necessary. I purified the concatamerization from low melting agarose.
As for cDNA elution from page gels, I would probably use some buffer, like TE or TBE, with the NaCl soln, but I never tried.