HELP!

Hello all! This is my first blogging attempt ever and I come to you in desperation! I have been attempting a simple cloning project and have had nothing but failure. I wonder if you have any suggestions for me? I am trying to clone a ~3 kb insert into a vector of ~9.8 kb containing CM resistance. The insert is engineered with terminal SpeI sites and has been cloned into Topo. Both constructs have been digested (either an hour or overnight) with SpeI, gel purified, and the gel purified product visualized on an agarose gel. I have phosphatase treated the vector with NEB’s Antarctic phosphatase (15-30 min at 37C) followed by heat inactivation (5 min at 65C, as recommended) or with NEB’s calf intestinal phosphatase (1 hr at 37C prior to gel purification). I have spec’d the DNA from the gel purification and set up ligations at 1:3, 1:10, and 3:1 (vector:insert, molar ratios). I ligate with NEB’s T4 DNA ligase overnight at 16C. I have tried various buffer stocks. I have transformed into house electrochemical comp cells after DNA precipitation. I have also tried direct transformation into Invitrogen’s Top10 chemical competent cells (30 min on ice, 30 sec at 42C). I transform between 10-40 ng/reaction in a volume of 25-50 ul cells. I have done outgrowths of 1-3 hours with normal (230 rpm) and reduced (150 rpm) shaking. The entire reaction is plated on CM plates and allowed to grow overnight (up to 48 hours) at 37C. When shorter phosphatase times are used with the Antarctic phosphatase, I get colonies on my vector alone. In all other conditions, I do not get a single colony. Any suggestions would be very much appreciated, as I am going crazy trying to figure out what is wrong in these reactions.


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