meth_4.1.5 Eukaryotic 18S V9 rRNA gene (1391F and EukBR) 1-step (5 PRIME HotMasterMix)

Contributor(s): Ramaciotti Centre for Genomics, UNSW, Sydney

The 18S protocol detailed here is designed to amplify eukaryotes broadly with a focus on microbial eukaryotic lineages. The primers are based on those of Amaral-­‐Zettler et al., (2009) and designed to be used with the Illumina platform. The protocol is based on that used by the Earth Microbiome Project (EMP), found here: http://www.earthmicrobiome.org/emp-­‐standard-­‐protocols/18s/

Primers for amplification: 18S V9 region (1391F and EukBR)

 

ILM_Euk_1391f PCR Primer Sequence – Forward primer

Field number (space-­‐delimited), description:

1. 5′ Illumina adapter
2. Index (indexed primer only)
3. Forward primer pad
4. Forward primer linker
5. Forward primer (1391f)

Non-indexed: AATGATACGGCGACCACCGAGATCTACAC TATCGCCGTT CG GTACACACCGCCCGTC

Indexed: AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TATCGCCGTT CG GTACACACCGCCCGTCG

ILM_EukBr PCR Primer Sequence – reverse primer

1. Reverse complement of 3′ Illumina adapter
2. Golay barcode*
3. Reverse primer pad
4. Reverse primer linker
5. Reverse primer (EukBr)

CAAGCAGAAGACGGCATACGAGAT XXXXXXXXXXXX AGTCAGTCAG CA TGATCCTTCTGCAGGTTCACCTAC

Full list of primer sequences can be requested from the sequencing facility.

 Preparation of master mix for amplification 18S V9 region (for 1 rxn)

(a)     PCR grade water was purchased from MoBio Laboratories (MoBio Labs: Item#17000-­‐11)

(b)     5 PRIME HotMasterMix (5 PRIME: Item# 2200410)

(c)     Final primer concentration of mastermix: 0.2 µM

Thermocycler conditions for amplification (for 96 well thermocycler)

Amplification method

• Use undiluted DNA as a first attempt, and 1:10 diluted for repeats/failed reactions. Please note that for the BASE project 1:10 was the default for the initial attempt
• Amplify samples with conditions outlined above.
• Run amplicons on an agarose gel. Expected band size for 18S V9 is approximately 200 bp.
• Clean and normalise samples in a one-step process using the SequalPrep Normalisation Plate Kit according to manufacturer instructions (Invitrogen Cat No. A10510-01).
• Combine equivalent volumes of normalised amplification into a single maximum-recovery tube.
• Perform a double cleanup of the pool using AMPure XP beads.
• Perform library QC on the pool using Qubit (concentration) and TapeStation (size). Calculate final molarity of the pool.
• Dilute pool to 4 nM.

 

Sequencing Primers

ILM_Euk_R1: Read 1 Sequencing Primer

Field description (space – delimited):

1. Forward primer pad
2. Forward primer linker
3. Forward primer

TATCGCCGTT CG GTACACACCGCCCGTC

ILM_Euk_R2: Read 2 Sequencing Primer

Field description (space – delimited):

1. Reverse primer pad
2. Reverse primer linker
3. Reverse primer

AGTCAGTCAG CA TGATCCTTCTGCAGGTTCACCTAC

ILM_Euk_INDEX: Index Read Sequencing Primer

Field description (space-­‐delimited):

1. Reverse complement of reverse primer
2. Reverse complement of reverse primer linker
3. Reverse complement of reverse primer pad

GTAGGTGAACCTGCAGAAGGATCA TG CTGACTGACT