Standort Bonn‐Köln th
UPDATED on 8 June 2013
Fon: 0228. 287-11055 Fax: 0228. 287-19144
[email protected]
RdRpSeq assay and NSeq assay RT-PCR for generating amplicons for sequencing targeting the Betacoronavirus (MERS-CoV) RdRp gene (RdRpSeq assay) and N gene (NSeq assay): Example formulation: Invitrogen SuperScriptIII OneStep RT-PCR System with Platinum® Taq DNA Polymerase First round
MasterMix: H2O (RNAse free) MgSO4(50mM) 2x Reaction mix* Fwd primer (10 µM) Rev primer (10 µM)
25µl single rxn, μl
SSIII/Taq EnzymeMix*
+ Template RNA
4.1 0.4 12.5 1 1 1 20 5
Professor Dr. med. Christian Drosten Director
Cycler: 50°C 20’ 94°C 3’ 94°C 15’’ 56°C 15’’ 72°C 30’’
45x
72°C 2’ ' = minutes; " = seconds
Dr. med. Jan Felix Drexler Staff Scientist Fon: 0228. 287-11697
[email protected] Victor Corman (Arzt) Staff Scientist Fon: 0228. 287-13185
[email protected] Dr. rer. nat. Marcel A. Müller Staff Scientist Fon: 0228. 287-13187
[email protected] Universitätsklinikum Bonn Sigmund-Freud-Str. 25 53105 Bonn
*Invitrogen: SuperScriptIII OneStep RT-PCR System with Platinum® Taq DNA Polymerase
Example formulation: Invitrogen Platinum® Taq DNA Polymerase Second round
MasterMix: H2O (RNAse free) 10xRx Buffer* MgCl2(50mM)* dNTPs(10mM each) Fwd-Primer(10 µM) Rev-Primer (10 µM) P.Taq Enzyme Template DNA received from the first round (µl)
50µl
Cycler:
single rxn, μl
36.8 5 2 1 2 2 0.2 49 1
94°C 3’ 94°C 15’’ 56°C 15’’ 45x 72°C 30’’ 72°C 2’ ' = minutes; " = seconds
* Invitrogen : Platinum® Taq DNA Polymerase
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Standort Bonn‐Köln Primers: RdRpSeq assay: EMC-SeqRdRP-Rev: EMC-SeqRdRP-Fwd: EMC-SeqRdRP-Rnest: W=A/T, N=A/C/T/G, R=A/G
GCA TWG CNC WGT CAC ACT TAG G TGC TAT WAG TGC TAA GAA TAG RGC CAC TTA GGR TAR TCC CAW CCC A
Fragment size: first round : 242bp (including primers), second round 228 bp (including primers)
Nseq assay: EMC-SeqN-Fwd EMC-SeqN-Rev EMC-SeqN-Fnest
CCT TCG GTA CAG TGG AGC CA GAT GGG GTT GCC AAA CAC AAA C TGA CCC AAA GAA TCC CAA CTA C
Fragment size: first round : 306-312 bp (including primers), second round 279-285 bp (including primers) Reference: Corman/Müller et al.; Eurosurveillance, Volume 17, Issue 49, 06 December 2012
Example pictures of gel electrophoresis of the resulting DNA of both assays:
RdRpSeq assay
NSeq assay
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