i isolated about 130 endophytic bacteria from medical plant, in identification step we have amplified 16sRNA gene for about 110 and sequenced them. BUT by using same primer, the rest of our isolates we try many times by increasing annealing temp based on the instructions of the company and we start from 55 to 58 and also changing the time and number of cycle but until now we can not get specific band as you can see in attached figure.
Dear Laurence Stuart Hall
Thank you very much for your kind answer and iam so sorry because i was thinking the figure attached but its not
in fcat i isolated about 130 endophytic bacteria from medical plant, in identification step we have done 16 s gene for about 110 and sequence them by using same primer, but the rest we try many times and following you suggestions such as increasing annealing temp based on the instructions of the company and we start from 55 to 58 and also changing the time and cycle but until now we can not get specific band as you can see in attached figure. Thank you very much once again
Andrey Luchnik
Thank you very much for your first answer, but the rest of your activity i think its not make any sense and also its not in right direction this is not Facebook or other social website. therefore, i suggest you to learn what is the right time and right place for such kind of these activity.
yes i work in china and i can speak Chinese,BUT iam egyptian. Russia and Egypt are very old friends and we respect Russian people. For Osama (ben Laden) i know you hate him because he was fighting with Russia but now he is deed and finish so he can not warns anyone.
finally, thank you very much for your first answer
Dear Osama,
I think you can try following things:
1. Change Mg2+ ion concentration. You can try concentration ranging in between 1.5mM to 3mM. This you can do while you change annealing temp. using a gradient thermal cycler. otherwise, change cation conc. by keeping the temp. constant.
2. You can use BSA or DMSO or glycerol in your reaction mixture. these act as macromolecular crowding agent.
3. Finally, are you getting this non-specific band problem for those bacterial cultures that produce polysaccharides ((i.e. mucoid colonies). If yes, you can either go for CTAB treatment during extraction of genomic DNA or you can can carry our purification of DNA from agarose gels. Precipitation behaviour of polysaccharides are very much similar with DNA.
Are you using 8-27f and 1492r primer set for amplification? if not you can try with this set. for sequence information you can refer to following article:
1. Saha & Chakrabarti. IJSEM 2006, 56: 991-995.
There are normally two major primer sets for bacterial 16S rRNA gene amplification, but 27f & 1492r set works best.
I hope it helps.
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