Oakey/Mijares Project

Purpose:

• Interrogating small artificial bacterial communities suspended in matrix to support controlled environment hydroponic agriculture at time intervals

  • closely related species, 16S too similar

  • RNA polymerase β subunit requested target

    • Hard to find or design universal primer for target

  • Proposed 16S-ITS-23S operon instead

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the  rrn operon

16S-27F and 23S-2241R

Fwd Primer to order: TTTCTGTTGGTGCTGATATTGCAGRGTTTGATYHTGGCTCAG

Rev Primer to order: ACTTGCCTGTCGCTCTATCTTCACCRCCCCAGTHAAACT

Barcoding 1700

PCR1 (target amplification):

“PCR mixture for the 16S-ITS-23S of the rrn operon (50 μl total volume) contained 5 ng of DNA template (or 2.5 μl of unquantifiable initial DNA), 1X Phusion® High Fidelity Buffer, 0.2 mM μl dNTPs 1 μM each primer and 1 U Phusion® Hot Start II Taq Polymerase. The PCR thermal profile consisted of an initial denaturation of 30 s at 98°C, followed by 25 cycles of 7 s at 98°C, 30 s at 59°C, 150 s at 72°C, and a final step of 10 min at 72°C.”

Add ul MM to each well

Cleanup (quant normalizing cleanup?)

PCR2(barcoding):

“PCR mixture for the barcoding PCR (100 μl total volume) contained 0.5 nM of the first PCR product (50 ng for the 16S rRNA gene and 142 ng for the 16S-ITS-23S), 1X Phusion® High Fidelity Buffer, 0.2 mM μl dNTPs, and 2 U Phusion® Hot Start II Taq Polymerase. Each PCR tube contained the DNA, the PCR mixture and 2 μl of the specific barcode. The PCR thermal profile consisted of an initial denaturation of 30 s at 98°C, followed by 15 cycles of 7 s at 98°C, 15 s at 62°C, 45 s (for the 16S rRNA gene) or 150 s (for rrn operon) at 72°C, and a final step of 10 min at 72°C.”

Add ul MM to each well

Add 2 ul of barcode and ul of the prior product

Cleanup (quant normalizing cleanup?)

References: