What could be the reasons for not getting colonies on transformation into E.coli competent after Side directed mutagenesis? There is no problem with DpnI digestion, competent cells, antibiotic used. In the same transformation the plasmid without mutation shows numerous colonies, so there isn't any problem with the transformation too.
I can also see the amplified plasmid after SDM-PCR on the gel.
We were having the same problem some time back but after troubleshooting we found that our competent cells were not that much efficient we changed our cells from xl to DH5 alpha and also that amount of pcr product used for transformation should be standardised as this is a bit toxic to cells.
In your case If you are saying that you visualised the pcr product on gel then I think there is not any problem in primer designing or pcr.
Could you please mention how many colonies you got after transforming DPN minus pcr product and in positive control how many colonies you mean by saying numerous??
in positive control has approx 200 colonies.
DpnI minus pcr product shows 2 or 3 colonies that too after 1 day (which peculiar as the competent cells are XL1 Blue E.coli)
I suggest you to change your competent cells because 200 cells in positive control is nt good enough. In our positive control in SDM we are getting uncountable colonies using 100 ng of DNA.
Good luck...
Ok fine..but there is be no problem if you will change your cells .....if possible use DH5alpha strain....
How many microlitres of pcr product you used for transformation?
Are you sure about the visualisation of pcr product in gel because usually it z difficult to get that visualisable amount of product as cycle number and template conc. In SDM are kept low...
And if you are getting just four colonies in DPN negative plate it points out that your initial template conc. Used for pcr is very low...how many ng of template you used in pcr?
ohk, i think that could be a problem, for 50ul, template amount = 2ng
PCR product after amplification of plasmid is coming out to be 0.6ng/ul. i.e. 30ng in 50ul. I transformed 7ul of this PCR product, 4.2 ng
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