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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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.

supplement0.docx
Table 1 Bt-CoV HKU5 Sequencing Primers
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Table 3 Bt-CoV HKU5 Amplicon Primer Sets
supplement0.doc
Table 2 SARS-CoV Sequencing Primers

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Bt-CoV HKU5 PCR/Sequencing Primers

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