The time you ran the gel is too low for the voltage you used. Try running at 100V for atleast 1 hour. Run more if still not properly separated.

Hi Sandu,

First please consider the bellow suggestions:

1. Dilute your digestion samples and also your ladder

2. Let your gel electrophoresis run further so bands separate completely

3. Increase the voltage to 90-100

4. Digest your self-ligated plasmid (without an insert)

5. Load undigested plasmid (harboring insert) as control

What is your plasmid and insert size? Do you have your expected insert released in the current gel? How long did you treat your recombinant plasmid with restriction enzyme (s)? did you digest it with a single enzyme or two? How many sites are there on the plasmid for enzyme (s) used? Are all the lanes representing the same insert and plasmid?

If ligation and subsequently digestion reactions are successful you expect two bands including your linearized plasmid and insert on gel, and if your cloning is not successful then you only see a single band on gel (linearized plasmid). In case you see other band patterns varying between the lanes would be because of unsuccessful digestion which might be due to short digestion time or missing of any other factors required for digestion.

Further, white colonies could be tricky and not really containing the desired fragments; if this is the case you do not see your insert band after digestion … performing colony PCR before digestion can help you to verify the ligation as well.

I think you have to prolong your digestion reaction to let your enzyme perform a complete cut and also to check out if other required factors such as temperature, DNA/enzyme concentration etc. are set to optimum.

I agree that the bands are not adequately resolved. Run the gel longer or perhaps move to a lower percentage of agarose.

Hi everyone,

I'm also dealing with a Problem and I would like to ask about your opinion...

My Vector Backbone (same Plasmid; but cut from different regions for different genes) is varying 3.3kb to 3.4 kb and my inserts sequences are 2.1kb, 2.3kb and 2.3kb.

I tried with;

-1:2, 1:3 ratios,

-used fresh Buffers,

-didnt expose DNA sequences to UV too Long while cutting from gel,

-after isolating DNA sequences from gels, i run them to validate sequences and they were correct too,

-DNA concentrations (Vector Backbone) were changing between 9.5 to 11.5 ng/µl (and I increased Vector DNA amount with insert DNA amount regarding to 1:2 and 1:3 ratios too..)

-At the end of the Ligation, samples were incubated at 16C for overnight +/-18-20hrs

-agar plates were prepared fresh,

I would like to hear your recommendations & ideas...

Cheers,

Z. Ilker

Zeki Ilker Kanbagli, you have not said what the problem is. Did you get no colonies? Did you get lots of colonies but none had inserts. Were the inserts wrong?

Hello Michael,

I’m sooo sorry, you are right! At the end, I didn’t get colonies...

Thanks in advance!

Z. Ilker

If you got no colonies, then the first thing I would check is your transformation. Do a control transformation with uncut plasmid, you should get nearly a lawn on your selection plate (some people think a few hundred colonies on a control transformation is good, but it should be tens of thousands.). If not the transformation, then check the ligation buffer and enzyme.

In other words, when the experiment fails be sure you do all the controls and check all the steps.

Before performing ligation, I‘ve already performed transformation and then did maxiprep with those plasmids (vector backbone and target genes which were in different plasmids) and I got thousands of colonies just like you mentioned...

Then, I did double and triple restriction (with respect to vector backbone and targeted genes in different plasmids), DNA isolation from gels, run those isolated DNA sequences on 1%agarose gel (for validating sequences) and finally ligations for each gene...

Should I perform transformation again?

(Ps. Another question popped up in my head while brainstorming, maybe I should try sequencing with these DNA sequences that I isolated from gels, what do you think?)

Waiting for your response and thanks in advance!

Cheers,

Z.Ilker

I would probably repeat the transformations or perhaps the ligations and transformations. If the fragment sizes are correct, I'm not sure there is much value in sequencing the DNA fragments you purified.

Zeki Ilker Kanbagli considering previous comments I think there could be some problem in ligation.

Could you let me know:

1. you are doing sticky or blunt end ligation?

2. How you prepared your insert fragment (i.e. directly using PCR product as an insert? or digested from plasmid? synthetic fragment?)

3. how about your insert concentration?

4. what is the total concentration of insert and vector you use in ligation reaction?