Bt-CoV HKU5 PCR/Sequencing Primers

doi:10.1038/protex.2014.023

Method Article

Bt-CoV HKU5 PCR/Sequencing Primers

https://doi.org/10.1038/protex.2014.023

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Version 1

posted 10 Jul, 2014

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This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of Bt-CoV HKU5 genomes.

Genetics

Microbiology

coronavirus

HKU5

genetic analysis

sequencing

This protocol details the steps, reagents, and conditions required to sequence Bt-CoV HKU5 genomes in the forward and reverse directions. The protocol begins with the RT-PCR step, assuming that BtCoV HKU5 RNA purified using a standard procedure (i.e., TRIzol extraction) has already been performed.

Invitrogen SuperScript III kit

dNTPS (10 mM)

Random Hexamers

RNasin (if desired)

Thermo Phusion PCR enzyme

Primers (2 mM stock – see Tables 1 and 2)

Agarose

1X TAE Buffer

Ethidium Bromide

70ºC water bath

55ºC water bath

Thermal cycler

Reverse Transcription:

In a 1.5-µL Eppendorf tube, add 1-4 µL RNA and 1 µL Random Hexamers.
Incubate at 70ºC for 5 min.
Place reaction briefly on ice and assemble the reverse transcription reaction using the kit-supplied reagents (a master mix can be made if multiple reactions are being run): 4 µL 5X First-Strand Buffer

2 µL DTT

1 µL SuperScript III reverse transcriptase

1 µL dNTPs

1 µL RNasin (if desired)

x µL H2O to 20 µL

Incubate at 55ºC for 45 min to 1 h.
Inactivate the reverse transcriptase at 70ºC for 15 min. Place reaction on ice after inactivation.
Proceed with PCR setup.

PCR (with Phusion PCR kit):

Assemble PCR reactions to generate amplicons according to those detailed in Table 3 (for whole-genome sequencing) or with any combination of forward and reverse primers from Tables 1 and 2.
PCR reaction setup: 2 µL First-strand template

1 µL Forward Primer

1 µL Reverse Primer

5 µL 10X HF Buffer

1 µL dNTPs

0.5 µL Phusion polymerase

x µL H2O to 50 µL

PCR reactions are run under standard PCR conditions: 98ºC 5 min

35 cycles of:

98ºC 15 sec

xºC for 30 sec*

72ºC for ~45 sec/kb

72ºC 10 min

8ºC Hold

Annealing temperature is primer-dependent, but for most SARS-CoV primers in Tables 1 and 2, annealing temperatures 52-55ºC will work.

Confirmation of PCR products and sequencing:

Run PCR products (5 µL/reaction) on a 0.8% agarose/1X TAE gel to verify PCR success.
Purify PCR products with PCR purification kit of choice (the Qiagen PCR Purification Kit works well).
PCR products can be diluted to 150-200 µL/reaction to ensure that enough product is present for assembling sequencing reactions.
Assemble sequencing reactions according to the primer/amplicon combinations outlined in Table 3..

Reverse transcription: 1-1.5 h

PCR: 2-4 h

Sequencing: facility-dependent

Primers have been designed to give clean, single-band PCR products. If multiple bands are detected, alternate annealing temperatures may be required. It may also be possible that alternate bands indicate multiple genome patterns at that locus.

None.

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Table 1 Bt-CoV HKU5 Sequencing Primers
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Table 3 Bt-CoV HKU5 Amplicon Primer Sets
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Table 2 SARS-CoV Sequencing Primers

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Version 1

posted 10 Jul, 2014

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Bt-CoV HKU5 PCR/Sequencing Primers

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Abstract

Introduction

Reagents

Equipment

Procedure

Timing

Anticipated Results

Competing Interests

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