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[rkay447]

Hoping someone here can help since no one in my lab seems to know what is happening or what I can do to fix the problem. I'm trying to ligate a 650 base insert into a 3.1kb vector. There are three ligations I need (GST, GST-domain and GST-mutated domain). The GST-domain inserts went in without any issues but the GST alone is being difficult. By the way, the domain on the GST is only 20 amino acids so there really isn't a significant difference in sizes.

I am using the same plasmid prep (digested, cip-treated and gel purified) for all three and know that it is fine since the other two ligations worked great. I did the pcr followed by gel purification of the GST insert which was the perfect size. Digest with XhoI/XbaI (same enzymes as for the others) and pcr column purification. Quick ligase for 15 minutes and transformation into novablue cells. Got only about 4 colonies initially which all turned out to be empty vector. Re-transformed the ligation reaction that sat out overnight on my bench which resulted in one colony on the vector control, about 50-60 on the ligation plates. Did restriction digest analysis on minipreps from colonies and got a 2.1kb insert (???). Decided to do a pcr on the miniprep DNA with the GST-specific primers used to make the insert and again got a 2.1kb insert. Turned around and ran a bit of the purified vector and insert used in the ligation and they looked perfect (3.1 vector, 650 base insert).

So where is this 2.1 band coming from??? The only idea I've gotten from labmates is multiple inserts but it's not even the right size for two or three and I would have seen multiple products in the pcr but all I got was one perfect 2.1kb band. Any ideas, anyone?? Please, this is the last experiment I need for publication and I'm supposed to start writing my thesis (and manuscript) in three weeks. HELP!

Edited by rkay447, 06 April 2009 - 07:22 PM.