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I've been transforming E. coli via heat shock in order to insert oligonucleotides (around 50 nt); however, none of my experiments have given positive results so far. I begin to question the efficiency of chemical transformation, especially for short DNA fragments. Is there such a notable difference between chemical and electro transformation?

EDIT: I'm checking for CRISPR array expansion, so my oligos have a protospacer length and structure. I'm using BL21(DE3) cells which have an IPTG-inducible T7 that I'm first transforming with a Cas1-Cas2 plasmid. Then, I induce the cells in order to express those proteins, make them competent, and transform then once again but with the oligos and an eGFP plasmid (for some sort of selection). To check for positive results, I'm amplifying the leader-repeat junction of the CRISPR array and compare its length (via electrophoresis) with a usual array that has no integrands.

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  • $\begingroup$ How do you do your chemical transformation? Do you make the cells yourself? Do you have a positive control? $\endgroup$
    – Chris
    Commented May 7, 2018 at 17:30
  • $\begingroup$ Yes, I make the cells competent myself. I do have a plasmid control for antibiotic selection which, on the other hand, is successfully transformed. $\endgroup$
    – alexbrt
    Commented May 7, 2018 at 17:33
  • $\begingroup$ In an allusion to Damir's answer below, how are you testing positive results? A plasmid with an antibiotic resistance gene is easy to test for. But 50 nucleotide oligos won't carry a useful reporter gene. $\endgroup$
    – user137
    Commented May 8, 2018 at 6:11
  • $\begingroup$ When transforming, I insert around 30 times more oligonucleotides (as mol) than plasmid, assuming that if the plasmid has entered the cell, the probability that some oligos have also entered is higher. Indeed, that's still pretty much luck, but testing hundreds of colonies and getting 0 results makes me believe that something's wrong. By the way, I'm checking for CRISPR array expansion, so for testing positive results, I'm comparing the amplicon length (which is the leader-repeat junction) via electrophoresis and after that, I'll be doing some digestions to confirm the presence of my oligos. $\endgroup$
    – alexbrt
    Commented May 8, 2018 at 6:21

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How do you detect a potenially positive transfection? Short fragments might have proper teriary structures which may make them behave differently than you would expect. Also, noncircular DNA and RNA is targeted much faster for degradation in most cells if the right head or tail signals are missing.

To your question: pulsed electroporation with well prepared bacteria (state, buffer,...) is usually 5-100 x more efficient than chemically competent bacteria.

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    $\begingroup$ See my anser above. Regarding oligo degradation (by endonucleases for example), I should mention that before transforming, I induce the expression of the necessary proteins for the CRISR adaptation stage. $\endgroup$
    – alexbrt
    Commented May 8, 2018 at 6:26

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