First, your wells are overloaded. There is too much material in each well, even the ladder. That alone can cause mobility differences between products because of different amounts of salt, etc. I would cut the amount of material you put in each well at the minimum in half and probably 5x.

Secondly, if you truly want to perform a direct size comparison between your controls and your new plasmid, you have to single digest them all. Just one enzyme, cut all the samples individually, run them on the gel next to a size ladder.

Did you linearize the plasmids before gel electrophoresis? Since supercoiled plasmid will have a tight structure, they will run differently than expected. The only way to get an exact size of a plasmid by agarose electrophoresis is to first linearize it using a restriction enzyme with a single cut site.

Yes, I did the double digestion (KpnI and EcoRI), there is fragment in right size but the vector size is between Tvector and pHannibal (image 3: ladder, pHannibal plasmid, 2 digested ligation, Tvector plasmid).

I did the ligation frequently with three of my genes but this result is always constant.

I'm confused what is happen?

I attached my gel images.

Best wishes,

Zahra

I always meet this problem. The most common reason is the quality of the ladder, so I suggest you run a plamisd which you have known its size as a control. Good luck to you!

Following on Matthew's comment, did you single-cut the plasmid prep before running it on the gel?

First, your wells are overloaded. There is too much material in each well, even the ladder. That alone can cause mobility differences between products because of different amounts of salt, etc. I would cut the amount of material you put in each well at the minimum in half and probably 5x.

Secondly, if you truly want to perform a direct size comparison between your controls and your new plasmid, you have to single digest them all. Just one enzyme, cut all the samples individually, run them on the gel next to a size ladder.

Thanks all,

The volume of my digestion reaction is always 30 microliter and I run it in one wall on the gel. 

No, I didn't single-cut the plasmid prep. I always do double digest to be sure the insert is in new vector. Is it better to do single digest?

Best regards,

Zahra

You need to single-digest a plasmid in order to measure its size, yes. Circular DNA, especially if supercoiled, doesn't run at predictable sizes. Molecular weight ladders are only valid for linear DNA.

So you'd want to do a single digest to get the overall size, and the double digest to see the fragmentation pattern.

i think nothing problem in your digestion and ligation process but you did mistake in transformation process like you was used host cell, may be contaminated or less efficiance and get different type colony in single plate. i had also same type problem but when i change the host cell then i got perfect result same ligation product.

best of luck

Abid Ali

Sequence your ligated plasmid.  Use a primer going in each directions to be sure that your sequence at the ligated sites is correct.  Once our insert ligated just before the promoter in the plasmid.  The more you sequence the more you will know what happened. Best, Merrill Hille

Hi Zahra

I do not understand why after transformation the construct you did a double digestion and run again on the agarose gel? If this is your way to confirm the successful of ligation, transformation. I would say this is really a long way and not a significant procedure to know.

What I normally do, after ligation, transformation, I do the following:

1- I pick a single colony from the transformed plate, suspended with 15 ul water. Then from that suspension I take couple of microliters, grow it on 5ml LB + antibiotic, leave it overnight to grow.

2- At the same time, the rest of suspension, I do colony pcr for it, using a forward primer bind to the vector before my gene and the reverse primer which bind to my gene. Run pcr if there is a pcr product on the gel this is mean you have a successful ligation.

3- If you get a pcr product then you can do a miniprep for your overnight culture, send it to sequencing to confirm there is no mutation.

Dear Zahra,

its a common problem which you have actully some time your comb cell contamination or less efficiancy showing thats why you get not good result.

so you first check your comb cell might be helpfull for you.

I am also facing the same problem. I did double digestion using SalI and NotI. Digestion is ok and after digestion, the vector (nearly 4kb) and insert (nearly 1kb) were ligated overnight. Then, I skip plating (because I failed in first time; too many small colonies on plate) and continue to overnight culture in LB broth. After plasmid isolation, I got the result as attach photo. It is larger than the expected one. I don't know what are these two bands. If you know, please share your suggestions. 1 kb marker was used.