I have done a TOPO cloning of a 450 bp fragment (amplified with Pfu Pol using forward primer designed with 5'CACC(ATG) and reverse primers having no 5' CACC) into pET101/D-TOPO vector in 1:1 and 1:2 insert:vector molar ratios as suggested by Invitogen.Upon transformation to TOP10 E. coli cells and plating on LB-Amp (50 ug/ml) plates, I see an efficiency of approx 3.5 x10 7 cfu/ml/ug DNA.I normally do a pre- screen using colony PCR with the PCR primers and those positive ones are later screened for orientation by plasmid isolation and RD. To my surprise I was getting less than 20% positive clones by colony PCR.But later I isolated plasmid from all of them and performed PCR to find that all of these answer PCR to a weaker extend compared to the colony PCR positive ones. Further I went on for digestion with XbaI (vector) and BamHI (insert).The expected results were a 150 bp release for the correct orientation and 350 bp for reverse orientation. But none of these PCR positive plasmids release any inserts. My positive control for digestion was a recombinant pET101D TOPO with an insert having BamHI site. It worked perfectly well with the digestion of positive control. What would one infer from this scenario? What might have happened during the ligation?
Dear Sai...Check your competent E.coli cells for plasmid contamination.
Dear Naveen,
Thanks for your suggestion. It is a good Point. I have done that already. They are commercial ones which comes along with the Champion pET TOPO expression kits and they are not contaminated. But your answer reminds me of possible contamination with the template plasmid used for PCR.I have used 1 ng/ ul template plasmid (again a TOPO plasmid with the full length gene) in 20 ul PCR reaction. But the post reaction the PCR mix was diluted X100 before quantifying the DNA content. So the possible concentration of template plasmid should be less than 1 pg/ul, against the equimolar concentration of PCR Product used with TOPO vector in ligation reaction ..Does this small amount of template gets transformed and produce colonies on LB-Amp plates ??.. What does the faint PCR response Vs Strong PCR response of equal concentrations of plasmids isolated from different clones suggest?? Why there is no response to the restriction digestion??.. I know it doesnot sound rational , but strange!!
You performed colony pcr by gene specific primer? ? If so, and you get positive colonies go for sequencing.
Hi Anwar,
Yes I have done with gene specific primers. I have almost all positive clones with only variation in the strength of the PCR Product. If I consider the strong ones as positive, why dont they answer the RD ??..I could get the answer by Sequencing, but this experiment is running as part of a course. So I need answer for why the RD let us down. Not responsponding to the restriction enzymes means either there is mutation at the sites or presence of some inhibitors of the restriction enzymes after plasmid preparation or this is a plasmid contamination. Even if it is the contamination by the template plasmid it has to give us answer for restriction digestion.So I dont find an answer to this at present
do restriction digestion of your positive clone by Xba1 and sac1 then see the result as i am not getting your point of Restriction digestion by xba1 and bamhi for orientation, i assumed that bamhi is in ur gene. i think for orientation of a gene in vector RD is not the best method. anyhow digest from other enzymes as i mentioned above and see the result. hope so you will get result.
First, only 20% positive clones dont make sense as gateway vectors contain ccdb gene which kills cells containing non-recombinant vector containing ccdb killer gene.
Secondly, you must do restriction digestion with some other enzymes also.
Hi Anwar,
You assumed it right; the BamHI is within the gene and XbaI in the vector and these two were successfuly used for assessing the orientation of insert previously. Meanwhile I was doing digestion with BamHI and HindIII and successfuly released my insert from the recombinant plasmid. I tried using BamHI and EcoRI (both XbaI and EcoRI are at the 5' end of TOPO cloning site and HindIII is at the 3' end), but failed to release the insert. What should I assume from such a result??..The distance between the restriction sites between BamHI and EcoRI or XbaI is 200 bp.In a poitive Control plasmid the distance is 480 bp. It is more than 350 between BamHI and HindIII sites. Does short distance between restriction sites create any hinderence for double digestion?? All the enzymes work well in single digestion and control reactions. Any idea why the RD fails in case of BamHI and XbaI !!
Miss Javaid, There was no mention about gateway cloning in my post and it is only directional TOPO cloning. Your second suggestion is welcome..
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