Help: in vitro peptide synthesis

Simonetta Palleschi spal at iss.it
Tue Aug 27 03:38:43 EST 2002


Hello to all of you!
Does anybody have experience with in vitro peptide synthesis? I'm using the
Promega TNT-T7 Quick coupled translation-transcription kit in order to
obtain the protein translation of PCR fragments from human genomic DNA (PTT,
protein truncation test; size fragments: about 1300 bp; gene: exon 11 of
BRCA1).

Each forward primer contains the T7 promoter sequence and a eukaryotic
translation initiation sequence. After synthesis, the products are analyzed
by 12% SDS-PAGE and autoradiography.

My problems:
1) the size of translated peptide it's larger than expected (about 65 kD vs
50 kD)
2) an unexpected,  high molecular weight peptide - about 85 kD  - appears on
the autoradiography

Any suggestion is welcome..

Thans,
Simonetta Palleschi





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