12.5 L 2x Taq Premix (contains enzyme, buffer, dNTPs) 4 L Primer 27f (5 M) - 5' –AGAGTTTGATCMTGGCTCAG - 3’ 4 L Primer 1492r (5 M) - 5' -TACGGYTACCTTGTTACGACTT – 3’ 3.5 L dH2O 3. Pipette 24 L master mix into each PCR tube, add 1 L of the appropriate DNA sample or sterile water (negative control). 4. Close tubes, load in thermal cycler, initiate thermal cycling program. Program = rRNA.fl Phase 1 (initial denaturation) - 1 cycle Initial denaturation 2 min. @ 94oC Phase 2 (standard cycle) 35 cycles standard denaturation 30 sec. @ 94oC Primer annealing 30 sec. @ 50oC Primer extension2 1.5 min @ 72oC Phase 3 (extra extension) - 1 cycle Primer extension 9 min. @ 72oC Day 2, Tuesday June 3, 2014 Session 1b - 3:00-5:00 – Download, Filter & Assemble Data G1c. Primary Assembly 1. Login to lab computer with userid: Guest2 pw: Biol0gyDept 2. Use Remote Desktop Connection to log into the Juniata GCAT-SEEK Server (192.112.102.20 ). Use the username and password provided to you (in computer lab, click “Use another account”). This server has 64 GB RAM, sufficient for a reasonably rapid assembly of prokaryotic genomes. 3. Use Google Chrome to visit the sequencing center download site and login with username and password provided to you. 4. Right click on the desired file, choose “save file as” and specify an appropriate download location (your folder on the data drive). 5. On the Start menu, choose 7-Zip File Manager, then browse to your files, select them, click the extract button, then OK. Close the 7-Zip File Manager. 6. Using Windows File Manager, move the uncompressed R2 file to the R1 folder, delete the R2 folder, and simplify the R1 folder name.
12.5 L 2x Taq Premix (contains enzyme, buffer, dNTPs) 4 L Primer 27f (5 M) - 5' –AGAGTTTGATCMTGGCTCAG - 3’ 4 L Primer 1492r (5 M) - 5' -TACGGYTACCTTGTTACGACTT – 3’ 3.5 L dH2O 3. Pipette 24 L master mix into each PCR tube, add 1 L of the appropriate DNA sample or sterile water (negative control). 4. Close tubes, load in thermal cycler, initiate thermal cycling program. Program = rRNA.fl Phase 1 (initial denaturation) - 1 cycle Initial denaturation 2 min. @ 94oC Phase 2 (standard cycle) 35 cycles standard denaturation 30 sec. @ 94oC Primer annealing 30 sec. @ 50oC Primer extension2 1.5 min @ 72oC Phase 3 (extra extension) - 1 cycle Primer extension 9 min. @ 72oC Day 2, Tuesday June 3, 2014 Session 1b - 3:00-5:00 – Download, Filter & Assemble Data G1c. Primary Assembly 1. Login to lab computer with userid: Guest2 pw: Biol0gyDept 2. Use Remote Desktop Connection to log into the Juniata GCAT-SEEK Server (192.112.102.20 ). Use the username and password provided to you (in computer lab, click “Use another account”). This server has 64 GB RAM, sufficient for a reasonably rapid assembly of prokaryotic genomes. 3. Use Google Chrome to visit the sequencing center download site and login with username and password provided to you. 4. Right click on the desired file, choose “save file as” and specify an appropriate download location (your folder on the data drive). 5. On the Start menu, choose 7-Zip File Manager, then browse to your files, select them, click the extract button, then OK. Close the 7-Zip File Manager. 6. Using Windows File Manager, move the uncompressed R2 file to the R1 folder, delete the R2 folder, and simplify the R1 folder name.
ProkaryoticGenomicsBreakoutSession2014.docx