Using the same protocol as above I did two more ligations, ligation 1 had a insert to vector molar ratio of 1:1 and ligation 2 had a molar ratio of 2:1. I mini-prepped those 10 colonies then digested the mini-prep DNA with Bgl II, the agarose gel of the digests showed that my insert was not there, I got a single band at 6000 bp which I am assuming is re-closed vector no insert. Coli, I got 10 colonies on LB/Amp plates. My first ligation I did a insert to vector molar ratio of 4:1, after transformation into in-lab-made competent Top 10 E. The insert was PCR amplified, cleaned using column method, and agarose gel checked to make sure the gene was there-it was and there was little to no background bands so I did NOT do a gel extraction-this should eliminate the EtBr/UV issue. So I have a MyH2 gene insert that is 5800 bp with a concentration of 40 ng/ul and a pET100/D-Topo vector with a concentration of 15-20 ng/ul. Could you please suggest me how should I proceed to make this complicated cloning successful? Any suggestions will be highly appreciated. I usually used T4 DNA from Promega but for this difficult cloning I also tried concentrated ligase from NEB but none gave me success. I have also tried ElectroSHOX Competent Cells from Bioline. Each insert is to be cloned separately into the plant binary vector (not both inserts in the same vector). I am using PacI for cloning the first insert (6650 bp) and SacII for cloning the 6498 bp insert. The main problem is that I don't get a colony in the plate (kanamycin). I've been trying to do this cloning for several months but have not had any success yet. Now, I am trying to clone the plant transformation vector which is about 7.2 kb. After several subcloning rounds, the size of the insert is now fairly big one is 6650 bp and the other is 6498 bp. I am generating a targeting construct for homologous recombination in plants.
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