pT7-7 cloning

Michael J. Tuvin mtuvin at
Tue Jul 11 10:11:29 EST 1995

Your "pT7-7 problem" is most likely related to the design of your PCR
primers. NdeI digestion requires quite a bit of an overhang on either side
of CATATG-palindrome. Recognition site provides for recognition but not 
necessarily is sufficient for the enzyme attachement. NEB catalog (1995) 
has a good table (p208) of a chain length required for the digestion with 
different enzymes. It will provide you with a reference point.
When I design my primers for the NdeI-add-on-PCR I provide for at least 12
nucleotides of some nonsense sticking out in front of the site.
Good luck

PS: If you happened to have the sequence of pT7-7 I would appreciate your 
sharing it with me (by e-mail). We have the vector itself but do not have
the sequence outside the cloning cassette.
Thank you
Michael J. Tuvim 
Research Assistant Professor, Dept.of Medicine, Baylor College of Medicine.
VAMC, bldg 109, rm 106, 2002 Holcombe blvd, Houston, TX, 77030
Tel: 713/794-7794(5), FAX: 713/794-7853, e-mail: mtuvin at

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