Table 2 of Yip, Mol Vis 2007; 13:2183-2193.


Table 2. Protein truncation test: primers for cDNA synthesis, first round polymerase chain reaction and second round polymerase chain reaction

Primer NR3 is used for both cDNA synthesis and the amplification of fragment CHM-2.1 in the first round PCR. Both primers CHM-1FT7myc and CHM-2FT7myc have a 5' tail, which has the following sequence: 5'-GGA TCC TAA TAC GAC TCA CTA TAG GAA CAG ACC ACC ATG G AAC AAA AAT TAA TAT CGG AAG AGG ATT TGA AT-3'. This tail has a T7 RNA polymerase promoter sequence at the 5' end (red color), a spacer sequence (7 bases), a consensus Kozak sequence (blue color), and a c-myc-tag sequence at the 3' end (green color). The promoter sequence facilitates the in vitro synthesis of RNA. The Kozak sequence facilitates the initiation of in vitro synthesis of protein. The c-myc-tag produces an N-terminal c-myc peptide (MEQKLISEEDLN) which facilitates the detection of translated polypeptide by anti-c-myc antibody.

                      Fragments                             Primers
                 --------------------   ----------------------------------------------
     Stage        Name        Size         Name         Sequence (5'-3')
--------------   -------   ----------   -----------   --------------------------------
cDNA synthesis    -        2102 bases   NR3           CCTTATAATTGCTGCTGCTTTCTAACA

1st round PCR    CHM-1.1   1500 bp      NF1           TAATAGTCACATGACACGTTTCCCG
                                        NR2           AATGACCCGAACAGCAAAAGTTCCT

                 CHM-2.1   1382 bp      NF2           TTAGTATCAAAGCTGCTGTATTCTCG
                                        NR3           CCTTATAATTGCTGCTGCTTTCTAACA

2nd round PCR    CHM-1.2   1524 bp      CHM-1FT7myc   Tail-GATACTCTCCCTTCGGAGTTTGAT
                                        CHM-2R        AGCAAAAGTTCCTGGTTCCTCTGCT

                CHM-2.2    1426 bp      CHM-2FT7myc   Tail-TCTCGAGGATTACTAATTGATCTTCTA
                                        CHM-3R        ATTGCTGCTGCTTTCTAACATTTCTCA

Yip, Mol Vis 2007; 13:2183-2193 <http://www.molvis.org/molvis/v13/a248/>
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