Dear Aarthy,

The first likely cause that I would think of is that one of your cloning reagents is contaminated with that 7 kbp insert. Next time I would try other, ideally fresh, reagents to be on the safe side. I do not know how you transform your E. coli but anyway make sure you do not inadvertantly contaminate your transformation solution at this stage.

In the past I have seen cross-department contaminations leading to cloning completely different inserts (whose origin became obvious only after sequencing and thanks to knowing what the other departments were working on at the time) when people from different departments used the same electroporator. To this day nobody knows how that was possible :-)

Good luck!

Kind regards,

Radek

Hi Aarthy,

I agree with Radek, there is cross contamination in your mix. Use all reagents in sterile working conditions. There are chances of getting contamination from pipettes and millQ water. Also check the constructs by DNA sequencing. I am sure you will get is solve.

Best wishes

Lokesh

Is your cloning directional? Are you cutting at the cloning sites, or a diagnostic internal site?... point being, maybe your fragment is just in backwards, so you would get a different sized fragment than expected when you digest (you didn't give much detail, so don't be offended... I have seen numerous students do this over the years!). Otherwise, you may have cloned what we refer to an "outer space sequence", i.e. nobody knows where it came from. Easy to do with PCR if you're not careful.

Hi Aarthy,

just wondering if the target for your PCR was a 7kb Amp resistant plasmid? So carry over from the PCR would be the cause of your problem. This is speculation, realy need more information as to what you have done.

-Richard 

Thank u ALL for ur suggestions and replies :)

The target for my PCR was a (5493 + 684 bp) Amp resistant plasmid... After PCR amplfn the target was purified by a PCR purifn kit.

Then the cloning of the PCR amplified fragment (684 bp ) was done in a pJET vector (2974 bp) following the manufacturers sticky end ligation protocol .... 

Aarthy,

Norbert's answer is the nice one I have read today. Apart from above recommendations of using fresh solutions, new competent cells,

1) after PCR purification, you can restrict digest and gel extract the insert and ligate into the final vector(i.e, avoiding pJET vector, since your product size is less). If you can do, use high fidelity polymerase and avoid UV exposure as much as possible. 

2) inferring the reason for your non specific require more info, as Richards referred.

3) use other plasmid extraction kit-solutions (Genomic DNA contamination)

4) Screen more plamids by colony PCR and gel run plasmids before restriction digestion(select Size~3kb for pJET vector+insert for digestion)

all the best

Hi Aarthy,

so your target plasmid is 6177bp, not quite 7kbp. Does it have the restriction site you are using? If not than it's possible you are seeing uncut vector running at 7kbp - easy to check against some uncut vector.

I note that in their protocol they specifically warn against carryover from Amp resistant plasmids. Gel purification no mater what size is useful to help remove this possibility. Or a DpnI digestion will also work to remove plasmid contamination, what ever the source. 

-Richard

https://tools.thermofisher.com/content/sfs/manuals/MAN0012707_CloneJET_PCR_Cloning_20rxn_UG.pdf

https://www.neb.com/products/r0176-dpni#tabselect0

When you are using a plasmid as template for PCR, you may get enough plasmid in the gel purified PCR product to transform number of E coli. You need to get rid of the template plasmid from your PCR product before going to ligation. Easiest and best way is to digest with DpnI.

Thank u ALL for your replies.  Will try out your suggestions.

Dr. Richard, thank you for the details.