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Cloning Oligos - (Oct/24/2006 )

Hi,
I am trying to clone a 78bp double stranded oligo. I have annealed the ds-oligo from synthetic oligos. The ds-oligo carries ends complimetary to the ends in the vector which are cut with Xho1 and Nhe1.
I got single colonies each time after ligation and transformation in TOP10 cells (phosphorylation and dephosphorylation combination- vector and insert).
When I isolated the plasmid (miniprep) I got more bands than my original plasmid and the most prominent band ran lower. I could not digest the plasmid with the restriction enzymes. ( only top two bands ran lower and brighter)
What is surprising is that 1)how the cells grow ? (in the colony and in the LB media)
2) the plasmid appears different (and smaller) than the original plasmid
I have attached the picture of the gel of the original and the new plasmid before and after digestion.

Please help me in making sense of all this and suggest possible ways out of this

Thank sad.gif

-checkmet-

Originally how big is the plasmid? and what the size you expect after double digestion?
did you have a control in your ligation of just the double digested plasmid to verify that you dont get self ligation due to incomplete digestion? (which I think might be happening)
your diestions and extractions are not very clean.
Sometime I got some colonies but end up extracting an X plasmid that gave resistance to amp.. for me that was contamination.. be sure you dont have this

QUOTE (checkmet @ Oct 24 2006, 06:35 PM)
Hi,
I am trying to clone a 78bp double stranded oligo. I have annealed the ds-oligo from synthetic oligos. The ds-oligo carries ends complimetary to the ends in the vector which are cut with Xho1 and Nhe1.
I got single colonies each time after ligation and transformation in TOP10 cells (phosphorylation and dephosphorylation combination- vector and insert).
When I isolated the plasmid (miniprep) I got more bands than my original plasmid and the most prominent band ran lower. I could not digest the plasmid with the restriction enzymes. ( only top two bands ran lower and brighter)
What is surprising is that 1)how the cells grow ? (in the colony and in the LB media)
2) the plasmid appears different (and smaller) than the original plasmid
I have attached the picture of the gel of the original and the new plasmid before and after digestion.

Please help me in making sense of all this and suggest possible ways out of this

Thank sad.gif

-tertu-

QUOTE (checkmet @ Oct 24 2006, 09:35 AM)
Hi,
I am trying to clone a 78bp double stranded oligo. I have annealed the ds-oligo from synthetic oligos. The ds-oligo carries ends complimetary to the ends in the vector which are cut with Xho1 and Nhe1.
I got single colonies each time after ligation and transformation in TOP10 cells (phosphorylation and dephosphorylation combination- vector and insert).
When I isolated the plasmid (miniprep) I got more bands than my original plasmid and the most prominent band ran lower. I could not digest the plasmid with the restriction enzymes. ( only top two bands ran lower and brighter)
What is surprising is that 1)how the cells grow ? (in the colony and in the LB media)
2) the plasmid appears different (and smaller) than the original plasmid
I have attached the picture of the gel of the original and the new plasmid before and after digestion.

Please help me in making sense of all this and suggest possible ways out of this

Thank sad.gif


Hi Tertu,
Thanks for the reply. My plasmid is 5.4 Kb and the previous insert was also 78 bp. So there is not much difference in the plasmid and the vector. Althogugh I can see the 78 bp fragment on the gel after double digestion.
I had negative controls and I am sure that the vector is fully cut by the enzymes.

-checkmet-