This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Bt-CoV HKU5 PCR/Sequencing Primers
Rachel Graham
ORFeome
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of Bt-CoV HKU5 genomes.
Keywords
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.
This is a list of supplementary files associated with this preprint. Click to download.
- BtCoVHKU5AmpliconPrimerSets.doc
Table 3 Bt-CoV HKU5 Amplicon Primer Sets
- HKU5.5Primers.docx
Table 1 Bt-CoV HKU5 Sequencing Primers
- SARSPrimers.doc
Table 2 SARS-CoV Sequencing Primers
Version History
CURRENT STATUS: Posted
Version 1
Posted 10 Jul, 2014
Metrics
Comments: 0
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Associated Publications
A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant
by S. Agnihothram, B. L. Yount, E. F. Donaldson, +16
Method Article
Bt-CoV HKU5 PCR/Sequencing Primers
Rachel Graham
ORFeome
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 10 Jul, 2014
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of Bt-CoV HKU5 genomes.
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.
Associated Publications
A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant
by S. Agnihothram, B. L. Yount, E. F. Donaldson, +16