I have a 4.8 kb vector plasmid and an insert sized 141 bp post restriction digestion (it is 151 bp post ligation and post restriction digestion it is 141 bp).
I have tried this multiple times, and I am having an issue with the ligation of the dephosphorylated vector and the insert.
For context- I use NEB Quick CIP for a 10-minute dephosphorylation and follow the 2 min 80 deg C deactivation step too. I do this only for my vector and not my insert.
I set up 2 ligation reactions- 1 with the dephosphorylated vector with the insert and 1 with the vector without dephosphorylation with the insert as a control- and I got colonies in the control reaction but no colonies in the other one. I used DH5 Alpha bacterial competent cells from NEB for transformation and followed the protocol for the same.
Additional info- I have double-checked both the PCR product and the digested DNA on 1% agarose gels and I use the Gel Purification Qiagen kit for purifying the DNA post running them on gels. So I can safely say there isn't any issue with these.
For the ligation reactions, I use a molar concentration ratio of 1:7 for the vector: insert, calculated from the NEB bio calculator based on the respective sizes and concentrations post digestion.
Please help me figure out how I can get the dephosphorylated digested plasmid and the insert to ligate and get colonies...
My colleague in the meantime suggested me to check for my gene of interest from a few of the colonies from my non-dephosphorylated vector-insert ligated control colonies, and perhaps there maybe some positive ones but most of them maybe the religated vector only since I use a single restriction enzyme for the digestion step...
Please let me know if any other information is required and I would be more than happy to provide the same, thank you so much!
Hello,
what's the restriction enzyme you are using ? blunt or cohesive ends ?
You said your insert is 141bp post ligation and 151bp postligation, what do you mean by that ?
I suppose you amplified your insert with primers containing the restriction site right ? Good practice would be to let at least a 6 nucleotide overhang at the 5' end of your primer for the restrcition enzyme to do its job correctly.
like so:
5'- ATGCAT-GGATCC-ATGCATGCATGCATGCAT-3'
overhang-restriction-prmier
For a true negative control, you need to transform bacteria with a ligation product only containing your opened backbone vector.
If, indeed you have more colony with the transformation of backbone+insert ligation than you have with the backbone alone, then you surely have some positive colonies.
As you pointed out, there is a good chance the colonies you saw are backbone religated on intself but it doesn't cost much to do some colony PCR, you may have some luck !
Hope it helps !
When you're gel extracting, are you illuminating with UV light? In that case I would guess you're not getting any colonies because of UV damage.
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