Manual isolation of plasmid i.e through ethanol precipitation always yields high as compare to kit isolation as quantity get compromised with quality i.e impurities but 30ng/ul is too low yield from kit isolation, I think u should take higher volume of overnight incubated culture 5-10 ml and after adding those lysis buffer solution and all pass it twice through column.. and during the final elution, after adding elution buffer/ sterile nuclease free water keep that column at room temp for few min before spin so that the complete elution buffer/water get soaked in the membrane filter of the column..

Thanx for the response Ravi Shankar Gautam.

The problem is not this actually since out of the 4 colonies only one yielded good concentration.

I have performed the instructions you have suggested (the initial colume of ON culture is 6 ml) and still it is the same. Somehow there is problem with these constucts and the problem might be the interaction of the plasmid with the guanidine based columns.

HOWEVER can you give any feed back about the no colonies formation even in the negative control (religation)?

What about bacterial transformation procedure? Which method did you use? Was it effective in that other plasmid (control) and control was performed with the same stock?

Thanx Angelika for the response!

The transformation worked really efficiently in the positive control (not related to my clonings of interest plasmid) but it did not work in the negative control (vector +YFP cut with one cutter / it is somehow no religated).

For some reason when the YFP is cloned in a vector the downstream clonings are not working at all!

George Rafael Samantsidis for no colonies their might be problem with your transformation procedure or ligation mixture reaction, u should try different ligation reaction concentration (vector and insert concentration) i.e 1:5, 1:7 1:9 (Vector: Insert), in case where insert size is comparatively too smaller than vector, insert should be taken in large amount i.e 1:5, 1:7 should be the ratio,,

Ravi Shankar Gautam how could you explain that there are no colonies in the religation of the vector without the insert. This is my question actually..

The YFP was subcloned into pSLfa vector with BamHI-NotI and then I took it with FseI_AscI cut sites in order to clone it into the kackbone vector. The problem is that when I cut this vector with AscI or FseI is linearized but no religated.

Georgios,

it is very hard to predict, I could only suggest. Problem could be in any downstream application. In my experience, plasmids with larger insertions could lose in transformation efficiency comparing to shorter control plasmids; and some other procedures (or chemicals) could be used as electroporation instead of the heat-shock. Competent E.coli cells are very sensitive and fresh culture preparation sometimes could resolve issues. You could try to use different stock of competent cells (in order if someone in the lab was used already this tube (or thawed it) and placed it back by accident). I agree with Ravi Shankar Gautam, about different vector:insert proportion. It is possible also to extend ligation time to overnight. With larger inserts I use to extend the growth time after transformation before plating. It is possible to concentrate cells before plating with gentle centrifugation. The interaction of cell-plasmid insert could be an issue usually if insert product is toxic to the cell, for example in case of cloning of an antimicrobial gene.

Do you have enough antibiotics on your plate? I would try also other bacterial strain. Do you have fresh plates. What concentration of Amp do you use?

Are you sure that AscI and FseI don't cut twice in your vector?

Agreed with Angelika Voronova...

Thanks guys for the suggestions but unfortunately there was no improvement and still after checking all the reagents with positive controls the problem is probably the column after gel purification.

Since I have tried to open five different preps of the vector with AscI (1 cut site in the vector) and after gel purification of these preps only one showed to be religated.

So the problem is probably the columns I am using for this purpose.

Even after phenol-chloroform purification of the prep after gel extraction the vector did not religate.