Enzyme Site in reverse Primer - (Nov/29/2006 )

I want to add protease site in reverse primer. The enterokinase enyme sequence is

5' GAC GAC GAC GAC AAG 3 '

to add it as a C-Terminal fusion

5' AAAAAA CTCGAG (protease site 3' CTG CTG CTG CTG TTC 5 ') TTCGGTGAACTTCCTGGATAGG 3'

I have to add its complementary sequence with the reverse primer.

3' CTG CTG CTG CTG TTC 5 '. Is it ??

or also reverse it

5' CTT GTC GTC GTC GTC 3'

or put it as such.

5' AAAAAA CTCGAG (protease site 3' CTG CTG CTG CTG TTC 5 ') TTCGGTGAACTTCCTGGATAGG 3'

is this your primer sequence!!!!? or the template?
if it is the template, u have to reverse it, no!
5' AAAAAA CTCGAG (protease site 5' CTT GTC GTC GTC GTC 3') TTCGGTGAACTTCCTGGATAGG 3'....so you can link your 5' to the free 3' end...correct me if this is wrong

then take the complementary sequence as your primer: 5' (protease site 3' GAA CAG CAG CAG CAG 5 ') ...

let us wait for other members....

5' AAAAAA CTCGAG GAC GAC GAC GAC AAG TTCGGTGAACTTCCTGGATAGG 3'
above sequence is my reverse primer sequence suppose and sequecne in red is the original sequence of the protease and i want to know that this sequece in reverese primer is come as such which i donot thinks so it is. either i have to add its compemenart sequence 3' CTG CTG CTG CTG TTC 5 'or compementary reverse sequence 5' CTT GTC GTC GTC GTC 3' to get the exact amino acid sequence in my peptide.

5' AAAAAA CTCGAG CTT GTC GTC GTC GTC TTCGGTGAACTTCCTGGATAGG 3' I am thinking this one can be the exact sequence for proease site in reverse primer.

hi
i think u are on track, ur reverse primer must be in the 'reverse complimentary' orientation which u have figured out i guess.....!

gud luk