Can anyone kindly help with the protocol(s) for 16S rRNA sequencing, cloning and expression of beta amylase gene in S. cerevisae. How to identify a microorganism using the 16S rRNA sequencing.
for the 16S rRna you need to do carry out PCR first using universal 16S primers that are easily available. If possible then make sure that you use 27F or 8F and 1391R or 1492R primers. The PCR reaction conditions are initial denaturation at 94 deg for 4 mins, 94 for 30 seconds, 56 or 55 for 1 mins, 72 for 1 min 40 seconds and final extension at 72 for 7 mins. once u get positive results go for purification of your pcr product. u can carry out peg purification (use 20 per peg of mol wt 8000). if positive u can send or carry out sanger sequencing for ur product (make sure for sequencing u use more at least three 16S rRna primers to get full length sequence). once u get ur sequencing results edit it by using chromas pro software and use EZTAXON database for identification.
for ur second question u can refer to sambrook and russel molecular cloning manual.