Hi, everybody, please help me. I met a strange ligation issue. I got the ligation product different to my expectation.

Here is the details:

insert: 1.1Kb (digested by BamHI and AatII from original plasmid or by PCR using infusion ligation primers)

vector: 8 Kb (digested by BamHI and AatII)

I can get colonies by T4 ligation and infusion ligation after transformation. However, all the extracted plasmids gave a size of 3 Kb. As shown in picture, 1-5 are extracted plasmids (single cleavaged by BamHI). Lane 6 is ladder, lane 7 is vector single cut by BamHI, lane 8 is vector cut by BamHI and then by AatII. Lane 9 is insert (by PCR with infusion ligation primers). The infusion primers were designed online. The insert and vector were purified by DNA gel and extracted by kit.

I already tried 10 times and picked up 40 colonies, it always give me this strange result.

Hope experts can save me.

Thank you very much!

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