README for MCPrimers.pm V2.0, mcprimers.pl, and CloningVector.pm 1. Authorship and copyright Author: Stephen G. Lenk, November 2005, May 2006. Copyright: Stephen G. Lenk (C) 2005, 2006. All rights reserved. This program is free software; you can redistribute it and/or modify it under the same terms as Perl itself. Licenced under the Artistic Licence. ############################################################################ # This software comes with no guarantee of usefulness. # Use at your own risk. # There is no guarantee this code will find the best solution, or even any # solution, or that the solutions it finds will be correct or useful to # you. # # Use at your risk. Check any solutions you obtain. # # Stephen G. Lenk assumes no responsibility for the use of this software. ############################################################################ Primer3 is called by this code to verify that the PCR primers are OK. Primer3 is Copyright (c) 1996,1997,1998,1999,2000,2001,2004 Whitehead Institute for Biomedical Research. All rights reserved. 2. Installation MCPrimers.pm - Place into Bio/MCPrimers.pm CloningVector.pm - Place into Bio/Data/Plasmid/CloningVector.pm mcprimers.pl - Front end script. Make available to users. PRIMER3DIR - Set this environment variable to point to diectory containing Primer3 executable. If PRIMER3DIR is not set, MCPrimers will set it to '.' by default. MSWindows - use primer3.exe Other OS - use primer3_core 3. Purpose Creates molecular cloning PCR primer pairs for a given gene so that the gene can be directionally inserted into a vector. pET-32a is the only vector currently supported. Solver is generic, restriction enzymes and their order in the vector are specified in mcprimer.pl and in CloningVector.pm 4. Use Use: perl mcprimers.pl [options] -vector name dna.fasta > result.pr3 Options: [-h] [-shift search_shift] [-clamp (both | 3prime)] -h help -vector cloning vector name (i.e. pET-32a) -shift integer value to shift search of left RE match into gene use -shift 12 (or other value) if initial search fails -clamp GC clamp 'both' will clamp last NT at both ends to G or C (default) '3prime' will clamp last NT on 3' ends to G or C Example: perl mcprimers.pl -vector pET-32a -shift 12 -clamp 3prime ppib.fa > ppib_all.pr3 Note: Use perl -Ilib ... if the modules are still in the local lib directory. Limitations: Does not account for redundancy codes in FASTA files. Note: These runs use intermediate files. This means that CGI or other use that can overwrite these files is a bad idea. 5. Test Results (V2.0) Passes test.pl clean on Windows XP with Activestate Perl 5.8.7 OSX with non-Activestate Perl (5.8.1) there are different results each time from mcprimers !? Passes test.pl clean on OSX with Activestate Perl 5.8.8 after data file was written in Unix format Passes test.pl clean on Linux with the standard installed Perl 5.8.7 using data file from OSX --------------- Enjoy, Steve Lenk slenk@emich.edu May 2006