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2
pCitrine
-3
TAA GGC CGG CCT GGA GGT GGA GGT GGA GCT
GTG AGC AAG GGC GAG
GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGC
CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC
CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA
CCTTCGGCTACGGCCTGATGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGA
CTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAG
GACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG
AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC
AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGA
ACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC
TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG
ACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG
ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GAC
GAG CTG TAC AAG
GAT CCT GCT GGT GCT GCT GCG GCC GCT GGG GCC AAA AGG
pCFP
-3
TAA GGC CGG CCT GGA GGT GGA GGT GGA GCT
GTG AGC AAG GGC GAG
GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGC
CACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC
CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA
CCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG
ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA
GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT
GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA
CAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAG
AACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAG
CTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC
GACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC
GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GAC
GAG CTG TAC AAG
GAT CCT GCT GGT GCT GCT GCG GCC GCT GGG GCC AAA AGG
Figure 2
. Nucleotide sequence of the Citrine-YFP and CFP tags contained in pCitrine-3 and pCFP plasmids,
respectively. Citrine-YFP/CFP sequences are indicated in blue. Yellow boxes indicate forward and reverse
primers for adding flanking linkers and restriction sites to Citrine-YFP/CFP (see Figure 1). Thick brown lines
indicate forward and reverse primers used to amplify Citrine-YFP/CFP for TT-PCR (see Figure 3).
Next, the fluorescent tag cDNA sequences are amplified from the pCitrine-3 and pCFP-3
plasmids using the Pfu-turbo DNA polymerase (Stratagene) and the forward and reverse Citrine-
YFP/CFP primers (Figure 3) to produce the “TT-Citrine” and “TT-CFP” fragments.
PCR reaction mixture
PCR cycles
100 ng DNA template
1x Pfu-turbo reaction buffer
0.2 mM 4 x dNTP
0.2 µM of each primer
0.025 U/µl Pfu-turbo (Stratagene)
total volume: 25 µl
1 cycle:
94°C
3 min
30 cycles:
94°C
30 sec
70°C
2 min
1 cycle:
70°C
2 min
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