another question sir. how can we find conserved regions of the 16 rRNA gene? do we need softwares for that?
Yes, Mark, you can use 16S primers for several bacterial species. What kind of bacteria are you working with?
THanks for the kind reply ma'am Milena. Im currently working with Halobacterial species ma'am. I am not yet confident to make my first 16s primer. any tips ma'am? do i need to consider the conserved regions? variable regions? (pardon for my innocence i'm relatively new on protocols ma'am. thank you! cheers.
yes some universal 16srRNA primers are available ..that u can use for several bacterial species
Hi Mark, I suggest you specify exactly what you are looking for as the primer you need depends on your application.
In general, if you just want to sequence 16s from one strain, go for the most common primers (say 8f-1492r) as a first guess and see if they work. IF you don't have them in your lab you can order from Sigma or some other company that have a catalog of common "universal" primers. If you are considerin something like qPCR you need to go for a shorter region in which case you have to be a little more careful but there are still plenty of primers to choose from. Things become harder when you are trying to selectively amplify one strain or group from a complex community, as you need to select a region that is conserved for this group but different from everyone else in the community. There you will need to do some more leg work, selecting a region, designing the primer and testing it in-silico before ordering it.
Good luck.
If you are not quite sure about exact bacteria then you may use Primer design softwares (may use NCBI tools, easy to use) by multiple alignment nucleotides of similar bacteria and can design primers based on conserved sequences and order them ... whereas, if you are looking for 16s rRNA, then they are highly conserved amongst bacteria and can just go through the cited literatures and order similar primers (like in your case look for Papers for Halobacteria). We use same approach, like recently we were looking for conserved genes for some biosurfactants so ordered similar primers which were already reported. Though I don't know exactly what aspect of Halobacteria you are working on, please find attached 1 thesis of one of my junior who worked on Halobacterium sp.- http://www.msubaroda.ac.in/phd2010/AparnaThesis17.pdf
Hope it may be helpful to you in some aspects. Best luck ...
Thank you everyone and sir Sanket for your help. I am now more confident to do my research! cheers!
Bear in mind that when designing primers a bit of mismatch at the 5' end of the primer isn't tragic; you really do want your 3' ends to be as conserved as possible amongst the species you will be working with (3' clamping )...
> how can we find conserved regions of the 16 rRNA gene?
> do we need softwares for that?
http://www.sciencedirect.com/science/article/pii/S0167701210002745
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