Ligation issues - (Jul/25/2005 )
Hi, I am trying to ligate a 40mer ds oligo into a 2.6kb vector. I have CIP treated my vector and phosphorylated my oligos before ligating with Quick ligase in 3:1 ratio. I then cut with a unique RE within the oligo and run on a gel.
My problem is that when I run the products on my gel, I seem to get a correct linear band (showing my oligo is inserted) but also seem to be getting an extra dimer band. It's a miniscule amount but my subsequent reactions are dependent on only having linear monomer!!
Why is this happening and how can I get rid of this extra band!! All help is greatly appreciated.
-chewie-
QUOTE (chewie @ Jul 25 2005, 03:03 AM)
Hi, I am trying to ligate a 40mer ds oligo into a 2.6kb vector. I have CIP treated my vector and phosphorylated my oligos before ligating with Quick ligase in 3:1 ratio. I then cut with a unique RE within the oligo and run on a gel.
My problem is that when I run the products on my gel, I seem to get a correct linear band (showing my oligo is inserted) but also seem to be getting an extra dimer band. It's a miniscule amount but my subsequent reactions are dependent on only having linear monomer!!
Why is this happening and how can I get rid of this extra band!! All help is greatly appreciated.
My problem is that when I run the products on my gel, I seem to get a correct linear band (showing my oligo is inserted) but also seem to be getting an extra dimer band. It's a miniscule amount but my subsequent reactions are dependent on only having linear monomer!!
Why is this happening and how can I get rid of this extra band!! All help is greatly appreciated.
oh,my god,i think you have gotten the very right result according to what you said above,the extra dimer band is your 40mer ds oligo which wasn't ligated into the vector but still remaining in your ligating reaction solution,if you wanna to get rid of it,maybe you can purify your product from gel.Best wishes!
-pfy1982-