Pgem t cloning kit

DNA was extracted from colorless filaments using DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. 16S rDNA were amplified using primers 8F/ 907R (for morphotype 1) and 8F/1492R (for morphotype 2) as previously described [38 (link), 39 ]. PCR amplifications were performed as follows: 95°C for 5 min, 35 cycles of 94°C 30 s, 58°C 45 s, 72°C 1min 30 sec and finally 72°C 7 min. PCR products were purified using QIAquick PCR purification Kit (Qiagen) and cloned with pGEM-T cloning kit (Promega) according to manufacturer’s instructions. Inserts from 20 positive clones of each construction were fully sequenced by Genoscreen (http://www.genoscreen.com) using vector primers T7 and SP6. The sequences obtained in this study were deposited in the GenBank database under accession no. KF892059 and KF892060.

Amplicons of 18S rRNA gene of medusa and polyp were directly sequenced by Genoscreen (http://www.genoscreen.com).
On the other hand, the prokaryotic 16S rRNA gene sequences were first purified using QIAquick PCR purification kit (Qiagen) and then cloned with pGEM-T cloning kit (promega) according to manufacturer’s instruction. Inserts from 13 positive clones were isolated and sequenced by Genoscreen (http://www.genoscreen.com) using the vector primers T7 and SP6. Following analysis, the 18S rRNA and 16S rRNA gene sequences obtained in this study were then deposited in the GenBank database under no. KJ493943 and KJ493944 respectively.