All primers were ordered by MWG (Ebersberg) and used at a standard concentration of 10 pmol/µl unless otherwise noted. The direction of the primers is indicated by S for sense primers and AS for antisense primers. All sequencing was done by MWG (Ebersberg).
C.5.1 Primers for SLAM and GAPDH amplification
To detect SLAM RNA expression in different cell lines of canine or simian origin respectively, the following primers were used.
C.5. Primers xxiii
Name/Target Sequence Melt.temp. Position in gene
canine SLAM S 5-TGGAAAGCAGGAGGGAAAATGA-3 51◦C 342 - 363
canine SLAM AS 5-TGAGGGCCGAGGCTGAGGTG-3 58◦C 602 -621
simian SLAM S 5-TCACTGGAGAACAGTGTCGA-3 50◦C 338,-,357
simian SLAM AS 5-CCAGCTGTAAGCCACATGGT-3 52◦C 642 - 661
GAPDH S 5-TGAAGGTCGGTGTGAACGGATTTGG-3 57◦C 5 - 29
GAPDH AS 5-ACGACATACTCAGCACCAGCATCAC-3 58◦C 258 - 282
C.5.2 Primers for sequencing
CDV-H and -F proteins were sequenced using standard sequencing primers binding to the vector upstream and downstream of the inserted gene. To sequence the middle part of the long proteins, primers binding to the middle parts of the genes were designed.
Name/Target Sequence Melt.temp. Position in gene
pCG S 5-CCT CTG CTA ACC ATG TTC AT-3 48◦C upstream of insert in pCG
pCG AS 5-CCA ACA CAC TAT TGC AAT G-3 45◦C downstream of
insert in pCG T71 5 -TAA TAC GAC TCA CTA TAG GG-3 46◦C upstream of insert
in pcDNA3.1
BGH rev1 5-TAG AAG GCA CAG TCG AGG-3 49◦C downstream
of insert in pcDNA3.1 CDV-H Seq1 S 5-AGC TAT TGC ATC GGC AGC-3 49◦C 483 - 500 CDV-H Seq2 S 5-CGG AGG AAG ACA GTT GCC-3 51◦C 1194 - 1211 CDV-F Seq1 S 5-GTC CTC GAA CCA ATC AAC C-3 49◦C 583 - 601 CDV-F Seq2 S 5-CTC CCT GTA TCC CAT GAG C-3 49◦C 1374 - 1392
1provided by MWG
xxiv C. Appendix: Protocols for molecular biology work
C.5.3 Primers for hybridisation
To tag CDV-proteins with the gene for the marker protein eGFP (enhanced green fluorescent protein), primers were designed having the recognition sites for convenient restriction enzymes to clone the hybridisation-protein into an plasmid appropriate for its expression. To reduce the possibility of a negative interaction between the CDV protein and the fluorescent protein, the two proteins were separated from each other by a spacer consisting of six alanines. The nu-cleotides for these six alanies also made up the overlapping region needed for the hybridisation-PCR.
Restriction enzyme recognition sites are printed in italics, start or stop codons in bold and overlapping sequences in small types.
C.5.3.1 Hybridisation of CDV-F with eGFP at its C-terminus
Name/Target Sequence Melt.temp. of
specific part
BamHI F S 5-TTAA GGA TCC ATG CAC AAG GGA ATC
CCC AAA AGC-3
55◦C
F 6Ala AS 5- cgc tgc ggc agc cgc tgc GAG TGA TCT CAC ATA GGA TTT CGA AG-3
54◦C
6Ala H/eGFP S tca ctc gca gcg gct gcc gca gcg ATG GTG AGC AAG GGC GAG GAG C
56◦C eGFP PmeI AS 5-TTAA GTTT AAAC TTACTT GTA CAG CTC
GTC CAT GCC-3
55◦C
C.5. Primers xxv
C.5.3.2 Hybridisation of CDV-M with eGFP at its C-terminus
Name/Target Sequence Melt.temp. of AAG ACC CTG ATC ATC GC-3
56◦C 6Ala eGFP S 5-gca gcg gct gcc gca gcg ATGGTG AGC AAG
GGC GAG GAG C-3
58◦C
eGFP ApaI AS 5-TTAA GGG CCC TTA CTT GTA CAG CTC GTC CAT GCC-3
55◦C
C.5.3.3 Hybridisation of CDV-M with eGFP at its N-terminus
Name/Target Sequence Melt.temp. of
specific part
KpnI eGFP S 5-TTAA GGT ACC ATG GTG AGC AAG GGC
GAG GAG C-3
58◦C
eGFP 6Ala AS 5-cgc tgc ggc agc cgc tgc CTT GTA CAG CTC GTC CAT GCC-3
54◦C
6Ala M S 5-gca gcg gct gcc gca gcg ATGACT GAG GTG TAC GAC TTC GAT C-3
55◦C
M ApaI AS 5-TTAA GGG CCC TTA GAG AAT TTT GAA
AAG ACC CTG ATC ATC GC-3
56◦C
xxvi C. Appendix: Protocols for molecular biology work
C.5.3.4 Hybridisation of CDV-H with eGFP at its C-terminus
Name/Target Sequence Melt.temp. of
specific part
BamHI H S 5-TTAA GGA TCC ATG CTC CCC TAC CAA
GAC AAG GTG G-3
58◦C
H 6Ala AS 5- cgc tgc ggc agc cgc tgc ACG TGG ACA TGA GAA TCT TAT ACG GAC-3
54◦C 6Ala F/eGFP S aac cgt gca gcg gct gcc gca gcg ATG GTG AGC
AAG GGC GAG GAG C
54◦C
eGFP PmeI AS same primer as for hybridisation with F
C.5.4 Hybridisation of CDV-H with eGFP following the transmembrane domain
Name/Target Sequence Melt.temp. of
specific part
BamHI H SPH 5-TTAA GGA TTC ATG CTC TCC TAC CAA
GAC AAG G-3
53◦C SPH 6Ala AS 5-cgc tgc ggc agc cgc tgc GAT AGC AAG CAA
GGC CAG GAT TC-3
55◦C
6Ala eGFP S 5-gca gcg gct gcc gca gcg ATG GTG AGC AAG GGC GAG GAG C-3
58◦C
eGFP 6Leu AS 5-gag tag aag cag gag tag CCT GTA CAG CTC GTC CAT GCC-3
56◦C 6Leu HR S 5-cta ctc ctg ctt cta ctc ACT GGA GTT CGA TTT
CAC CAA GTA TC-3
54◦C
HR PmeI AS 5-TTAAGTTT AAACTTAACG GTT ACA TGA GAA TCT TAT ACG GAC-3
54◦C
C.6. PCR-Master-Mixes xxvii
C.6 PCR-Master-Mixes
C.6.1 Reverse transcription of SLAM and GAPDH mRNA
MM 1 6 x
DEPC-treated water 60µl
dNTP[2.5 mM] 48µl
RT-buffer (5 x) 48µl
P 156µl RNA template per 26µl-reaction 6µl
MM 2 6 x
Dithiothreitol[0.1 M] 21µl random hexamers (1:15) 12µl
DEPC-treated water 3µl
RNase inhibitor 3µl
M-MLV reverse transcriptase[200 U/µl] 9µl
P 48µl
Addition to MM 1 per reaction 8µl
C.6.2 Amplification of SLAM and GAPDH cDNA
6 x DEPC-treated water 6µl Master Mix (Abgene) 270µl Primer S[100 pmol/µl] 3µl Primer AS[100 pmol/µl] 3µl P 282µl template per 47µl reaction 3µl
xxviii C. Appendix: Protocols for molecular biology work
C.6.3 Amplification of CDV proteins and hybridisation products
2 x DEPC-treated water 51µl Pfu-buffer with MgSO4, 10 x 10µl
dNTP[2.5 mM] 8µl
Primer S[10 pmol/µl] 10µl Primer AS[10 pmol/µl] 10µl Pfu-polymerase[2.5 U/µl] 1µl
P 90µl template per 45µl reaction 5µl
C.6.4 Colony-PCR
20 x DEPC-treated water 153µl
Tth-buffer, 20 x 10µl
dNTP[2.5 mM] 16µl
MgCl2 [25 mM] 10µl
Primer S[10 pmol/µl] 5µl Primer AS[10 pmol/µl] 5µl Tth-polymerase[5 U/µl] 1µl P 200µl
template per 10µl reaction one pipette tip colony