Trouble cloning miRNAs - (Jun/06/2007 )
Hello,
I'm trying to clone specific miRNAs via the Bartel protocol using the 3' adaptor ligation and RT part only. I'm trying to see if different nucleotides are post-transcriptionally added to the 3' ends of miRNAs so I am using 5' primers specific to chosen miRNA targets for PCR. The PCR products are run out on 15% PAGE gels and then stained with sybr green. I attached a gel with lanes 1, 2, 3, and 4 and then two -RT control lanes. Lanes 1 and 2 use a 5' primer 3 nt longer for pre-mir16 than the primer used for the mature mir16 shown in lanes 3 and 4. It appears that specific amplification is taking place in all lanes but I'm still getting a similar sized product in the -RT control lane. Does anyone have any idea what this product(s) could be? Also, what is the best way to isolate the amplified products from the gel to TA clone? Thanks in advance.
Mateo
couldn't it be just contamination from the next well? Did you try to leave an empty lane between samples and controls?
to clone the bands I would elute them overnight in 0.3 M NaCl, precipitate with 3 vol of ethanol , incubate with taq and dNTPs to make sure there are overhanging A and perform the T/A ligation
to clone the bands I would elute them overnight in 0.3 M NaCl, precipitate with 3 vol of ethanol , incubate with taq and dNTPs to make sure there are overhanging A and perform the T/A ligation
andrea, thanks for the comment. there was no contamination from well to well so I am really clueless as to how I could get any amplification in the -RT control lanes. do you think it would be worthwhile to clone the bands or just try the procedure again with higher temps for the RT reaction and PCR? thanks,
mateo
-RT means that you add no reverse transcriotase but polymerase yes? In this case you could be facing DNA contamination. Try a DNAse digest with th miRNA preparation and the do your RT-PCR...
Stardust
I would proceed with the cloning and see. Remember to use native (non-denaturing) PAGE.
Bye
Bye
I received sequence information from the cloned PCR products and they were indeed mir16 ligated to the adaptor. I'm happy that it worked but still curious as to what could have been amplified from the -RT control. Regardless, thanks for the help.
Mateo