enzyme site after blunting - (May/15/2007 )
i am very sorry to ask you, because i am very confused in this matter
if i blunt EcoRI site of a vector and KpnI site of an insert and after ligation of these two fragment , does EcoRI site will regenerate???
sorry i cant understand............
sequence of EcoRI site is GAATTC ( 5'-AATT) and after blunting it will be AATTC.
sequence of KpnI is GGTACC (GTAC-3') and after blunting it will be C only.
so after ligation the site will be AATTCC and this is not EcoRI site. am i right????
so you cut with Ecor1 ang get this:
GAATTC -> G + AATTC
CTTAAG CTTAA G
then you blunt it, so you get this:
G + AATTC -> G + C
CTTAA G C G
The same goes for Kpn1, no matter what the original site was,
You just basically loose the sites when you blunt them.
and better dephosphorylate your vector before ligation so that you don't recircularise it.
thanks for you answer but i didnot mean it in my question.
actually i want recircularization.
so i blunt both EcoRI and and KpnI and ligate to get recircularization.
and my question was what will happen after blunted end of EcoRI and KpnI will join,
is there any chance to in the new sequence (of blunted EcoRI and KpnI ) regenerate a EcoRI site again???
becuase when i check my plasmid i get ecoRI site in the blunted position but i am confused about the site after blunting
EDIT: Ignore this post. Error in KpnI sequence
My apologies for the crudeness of this drawing. But I hope it will better help illustrate the point that minus was making.
5' G | AATTCXXXXXXXXXXXCTTAA | G 3'
3' CTTAA | GXXXXXXXXXXXG | AATTC 5'
Blunt ending (Klenow)
5' GXXXX G 3'
3' CTTAA C 5'
5' GAATT G 5'
3' CTTAA C 3'
This sequence is not EcoRI. DNA's innate 5' - 3' orientation prevents ligation in any other orientation.
please check out my attached file
Ummm this is bad. I've made a mistake. I'm sorry. NEB says KpnI site is GGTAC|C
not CTTAA|G as I mentioned.
In which case
5' G | AATTCXXXXXXXXXXXGGTAC | C 3'
3' CTTAA | GXXXXXXXXXXXC | CTAGG 5'
Blunt ending (Klenow with 3'-5'exonuclease activity)
5' GXXXX C 3'
3' CTTAA G 5'
5' GAATT C 5'
3' CTTAA G 3'
which regenerates the EcoRI site
very sorry for the mix up with the KpnI restriction site sequence.
The 5'-3' standedness of DNA prevents any other outcome. So EcoRI will regenerate.
yessssssssssssss, that i wanted to say
yessssssssssssss, then i have the right plasmid