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Hello, I have a related question. I try to ligate annealed oligos into double digested (different enzymes) plasmid. The oligos I annealed were not modified with phosphates. Do I need to phosphorylate or dephosphorylate any of the two? As far as ...

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... to amplify 4 overlapping regions of the gene, run them on a gel, cut out the bands of expected size, purify them, and eventually sequence and ligate them together to generate the full length gene which I would then clone and amplify. However, there were troubles with the isolation of the bands. ...

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also sometimes when i ligate short oligos, i use different inster:vector ratios into different plates just to see the pattern.. hope it helps too!

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... into some chemically competent E. coli cells. I was wondering what I should run as a negative control? I know if the cloning works the plasmid ligates and an antibiotic resistant gene on the plasmid starts to be expressed. If E. coli cells take up the plasmid they will obviously grow on the ...

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... amount of splicing intermediates. I have so far used varying primers on the cDNA of arabidopsis in order to get some fragments that I could then ligate together. I have used proofreading polymerase, Taq, phusion, abgene, and I have then tried (and failed) to isolate, purify and sequence the fragment. ...

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