3 replies to this topic

[fabiola]

Hi,

I have designed primers to introduce new restriction sites to a vector to be able to introduce my cDNA, and I would appreciate it if anyone could check them and suggest any changes or confirm if it is correct.

Thanks.



Forward:

LANDING SITE [ MLUI ] Kozak Fragment [START] *GENE*

5’- GCGC [ACGCGT ] GCCACC [ATG] gcgggcagtagccgct -3’


Reverse:

LANDING SITE [BAMHI] [STOP] *GENE*

3’- CTAG [GGATCC] [CTA] ccacatttattctacact -5’

[bob1]

My only comment is that you need to put the add-ons on the 5' end of the reverse primer always! You could copy and paste what you have written above into a primer ordering site and it would give you the correct oligo at the end, it's just that you have written the 5' and 3' at the wrong ends.

[fabiola]

Thanks a lot for the reply...
What do you mean by the "add-ons"?

[bob1]

The restriction sites and other flanking bits.