introducing restriction site upstream of Kozak by PCR

Student mucineer at iname.com
Thu Jun 15 06:16:18 EST 2000


I tried repeatedly using an overhanging primer to introduce a restriction
site (AAGCTT) upstream of a Kozak sequence in the gene I want to express by
PCR. My primer has a 15-bp perfect match from the Kozak (GCC GCC ACC....
15nts in total) and has the "AAGCTT" immediately 5' of this, plus about 6
bps 5' of this restriction sequence just to give the enzyme some space to
work with.

This was done in a PCR with a reverse primer and then the PCR product was
TA-cloned.

What I found is that the restriction sequence simply disappeared in all the
clones I sequenced and is replaced by something unknown to me, the bit from
"GCC GCC ACC...." are correct but no restriction sequence was present
upstream of that.

Does anyone know what might be the problem? I 've done this type of PCR many
times before with other sequences and they all worked out fine...

Thanks.








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