Hi,

Have you transformed with your buffers alone or 'mock' clean-up reagents (do a purification without DNA)? This looks suspiciously like a common cloning plasmid like BlueScript or PCRScript that may suggest your purification reagents may be contaminated.

Is PCR product band purified? If not does template have the have same antibiotic resistance as target plasmid? Either gel-purify (under Blue light NOT UV!) or DpnI treat PCR product before clean-up. Gel-purification may be better if you also see evidence of primer dimers (perhaps not here).

Check your AatII In-Fusion primer and the In-Fusion design rules as AatII gives a 3' over-hang so requires additional bases in the primer for re-constitution of the site.

Do you have target vector only as a control transformation? it may just be that your target plasmid is not well digested and you may have to re-cut and purify it.

Good Luck!

Hi Nan Yang, indeed, as Nick says, it looks suspicious, your minipreps clearly yield something different from the input material, and with a reproducibility one would so much like for good results!

So, first I agree with Nick, does the 3 kb size comply with the plasmid you use to produce your 1.1 kb insert, whether by digestion or PCR? The insert needs to be purified by gel extraction, but even in that case, if your original plasmid is only 3 kbs you might easily get drag-over contamination, and few pg of undigested plasmid you will never spot on gel, but will give you tens of colonies in transformation if your cells have a standard 10E8 transformation efficiency. So, if you amplify by PCR, a DpnI digestion before loading on gel, as suggested by Nick, is a very good trick: DpnI digests methylated DNA which is of course only the one extracted from bacteria, so the plasmid, but not the PCR fragment. If you digest your fragment, I would suggest purifying on a 0.8% gel with a very slow run, maybe 40 mA of current in total, to maximize separation of the bands.

Secondly, it would be interesting to transform a control ligation or InFusion reaction where you put the vector only. This will give you an idea of how much single digested vector you have there, if you don't dephosphorylate. I don't know how close the BamHI and the AatII are on the vector, but it is possible that after BamHI cuts, then AatII cannot cut, possibly because the site ends up too much on the end of the DNA. It might be useful to cut first with AatII and then with BamHI which is a much easy-going RE (so the opposite of what you show in the gel)! Also for the PCR fragment, some enzymes really need space to bind to the DNA and cut, therefore it is advisable to have at least 4 nt after the restriction site, and 5 is better when it's a rare RE!

Third, I'm not sure what you mean when you say that the InFusion primers were designed on-line. The InFusion primers must have 15 nt of homology with the plasmid sequence after the cut on their 5' side, this you have to take care yourself, manually. Of course the 12-15 nt on the 3' side that amplify your fragment can be chosen with a tool if you don't have specific requirements (for example they include the beginning of a CDS and must contain a Kozak consensus and the ATG, or must overlap with the STOP codon, and so on...). So be sure your primers are well designed. Keep in mind that the InFusion system contains an exonuclease that will digest your DNA fragment in a 3' to 5' direction to produce a long (10-15 nt) 5' overhang of the opposite strand. This will anneal with the complementary overhang produced on the vector. So the design of the primers is very specific for the approach you use (single or double digestion, reconstitution or not of the REs, 5' or 3' overhang of the REs to obtain the appropriate homology length...).

Good luck!

Thank you both very much Nick and Pietro!

There are 15 nt between the double digest sites on the vector. I also tried the single-single digest to get the vector: BamHI digested overnight then purified by PCR column, and then single digested by AatII and then purified on DNA gel.

I got my insert from a plasmid (3.5 kb in size) by digest or PCR. The insert was purified on DNA gel and I only can see a single band. However, as both of you said I also think that the colonies may come from the insert plasmid.The problem is I also tried to sequence the plasmid that I got from ligation. I used 3 primers, one at the beginning of insert, one in the middle of insert and the 3rd one is in the end of insert. I got the results yesterday, there is only noise signal. It seems that there is no insert gene in my ligation plasmid. That is very strange. :(

DpnI digest is really a very smart idea, I will definitely do it. 

Now, I changed the cleavage sites on the vector and did the single-single digest again on vector and purification on DNA gel. Based on these two new cleavage sites, I also got new infusion primers for insert and did PCR and got my insert (single band) purified on DNA gel too. I did infusion ligation again and got many tiny colonies on agar plate. Today, I did the colony PCR (using the new infusion ligation primers) and all 4 picked colonies gave the correct size of the insert (1.1 kb). I will get plasmid tomorrow and will do single and double digest again to see if I can get the correct plasmid.

I sincerely appreciate your help!

I changed the cloning sites for ligation. It works now. I got correct plasmid sequence. Thank you all my friends!