too much background in vector preps - Vector prep (Oct/16/2007 )
In the past I have been successful in prepping vector , pUC18 and pMCL200, with very little background. But now it seems I have a lot of white colonies with no insert. Bad SAP dephosphorylation, should not be the problem because the colonies should turn blue. Both vectors have the lacZ gene and I am plating on bioassays with IPTG and X gal. Could this be a problem with the components of the bioassay? the comp cells? I have a negative control ligation, no DNA no vector, I did not transform and plate because nothing ever grows. I will try this tonight.
Does anyone have any suggestion to help me trouble shoot? Any low background vector suggestions?
Someone suggested degraded antibiotic might cause the problem, so I am plating on new plates plus adding extra antibiotic to some and see if this helps reduce the background.
Day2;
Still need help. I plated again with fresh antibiotic and double fresh antibiotic and I am still getting a lot of background.
When I transform ligation with no vector and no dna I get no growth, so I have eliminated the comp cells as the source of the background.
Can I transform linear vector that confers antibiotic resistance to the Ecoli? Could this be the cause of the background, if so what can I do to reduce it?
Does anyone have any suggestion to help me trouble shoot? Any low background vector suggestions?
Someone suggested degraded antibiotic might cause the problem, so I am plating on new plates plus adding extra antibiotic to some and see if this helps reduce the background.
Try sequencing a couple of clones. you might have a tiny fragment cloned in the vector.
could you give more infromation about the insert and the ligation stratrgy? More info would help. Sometimes dephosphorylation is more trouble then it is worth. If the ends are uncompatible, there is no absolute need for dephosphorylation.
I am making random shotgun small insert libraries. Hydroshear -> blunt end repair -> agarose size selection -> gel purification -> ligation
I have used epicentre fast link ligation and an overnight t4 polymerase ligation.
My negative control has no insert DNA and about 100 ng of pUC.
Does anyone have any suggestion to help me trouble shoot? Any low background vector suggestions?
Someone suggested degraded antibiotic might cause the problem, so I am plating on new plates plus adding extra antibiotic to some and see if this helps reduce the background.
Try sequencing a couple of clones. you might have a tiny fragment cloned in the vector.
I get the background when I add water to the ligation as a negative control, plus a do a pcr qc on my clones that reveals no insert.
Does anyone have any suggestion to help me trouble shoot? Any low background vector suggestions?
Someone suggested degraded antibiotic might cause the problem, so I am plating on new plates plus adding extra antibiotic to some and see if this helps reduce the background.
Try sequencing a couple of clones. you might have a tiny fragment cloned in the vector.
I get the background when I add water to the ligation as a negative control, plus a do a pcr qc on my clones that reveals no insert.
This makes me think there might be a problem with the plasmid itself. How old is the current prep of plasmid? You might need t o get a fresh source of DNA.
Does anyone have any suggestion to help me trouble shoot? Any low background vector suggestions?
Someone suggested degraded antibiotic might cause the problem, so I am plating on new plates plus adding extra antibiotic to some and see if this helps reduce the background.
Try sequencing a couple of clones. you might have a tiny fragment cloned in the vector.
I get the background when I add water to the ligation as a negative control, plus a do a pcr qc on my clones that reveals no insert.
This makes me think there might be a problem with the plasmid itself. How old is the current prep of plasmid? You might need t o get a fresh source of DNA.
The purified pUC18 plasmid was ordered recently. So it should not have been a problem with the vector. Nonetheless, I have ordered more and I am going to start over.