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Ligation gel picture interpretation - (Feb/01/2012 )

Hello, I am trying to insert a 2 kb PCR fragment with EcoRI/XbaI sites into a pET32a vector (5.5 kb) that is digested with EcoRI/XbaI. I did a 3 hour ligation.

Control: Vector + ligase - - - - This gave approximately 50 colonies
1:3 Vector:Insert - - - - - - again around 50 colonies
1:8 Vector:Insert - - - - - around 60 colonies

I am worried about all of the self ligation that occurred and I doubt that any of my colonies on experimental plates are the clone I am looking for.

I ran some of the leftover ligation on a gel and would like some input on the multiple bands I observe in the vector + insert ligation reactions. I will be doing minipreps and XbaI/EcoRI digestions tomorrow so I will find out then if it worked for sure, but is the expected 7.5 kb vector+insert band in the smear?

lane 1: marker (12000, 8000, 6000, 5000, 4000...)
lane 2: control (vector + ligase)
lane 3: 1:3 vector:insert
lane 4: 1:8 vector:insert

Also, if you have any suggestions to limit self-ligation since my control plate had as many colonies as the actual ligation they would be appreciated. I have not used SAP or the likes for the vector since I have incompatible ends.

Thanks in advance
Attached Image


the digest doesn't seem to be complete. Try to digest it completely and run the digest longer on gel before purifying it for ligation.


I digested the vector overnight (~18 hours) and it seemed to give a very defined band on the gel that I excised. Are you saying since there are three bands in the vector + ligase (lane 2) the digest of the vector must have been incomplete? I was thinking the top band could possibly be a dimer of the vector


if digested completely, you should have one clear band for the vector alone control group.


UPDATE: It turned out I did have clones from this ligation reaction, but as scolix predicted the majority were uncut plasmid that was transformed. I screened 18 colonies and I had 3 positive clones.