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In my group we recently tried to express a protein necessary to perform an analytical method in BL21(DE3). The first two attempts did fail and no colonies did grow on the agarose. I hear, that the extraction of plasmid from paper and resoubilization is more prone to error in comparison to plasmid that is shipped in liquid form and this is a known issue. I am wondering what could be possible workarounds to try to still get it working. Until know, I have the following ideas:

  • Increase plasmid concentration
  • Decrease antibiotic concentration
  • Use a different strain like NEB 5 alpha

I am also wondering if the X-Ray treatment of customes service could facilitate in any way plasmid determination or is this an urban myth?

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    $\begingroup$ How did you prepare the plasmid from the paper? Did you do a transformation and a mini prep first? $\endgroup$
    – Chris
    Nov 15, 2023 at 10:28
  • $\begingroup$ No miniprep. I took the sheet of paper and cut it leaving a small portion as backup. Solubilzed the plasmid with 50 microliter TE buffer, resulting in a plasmid concentration of 11 ng/ml. Using 4 microliter plasmid with 25 microliter bacteria culture. Did not work. Than increased to 10 microliter plasmid, did not work. I observed now increased plasmid concentration of ca. 17 ng/ml after adding 100 DNSase free water destilled water and letting it stand over night. $\endgroup$
    – raptorlane
    Nov 15, 2023 at 10:43
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    $\begingroup$ Ok: My advice in these cases would be to first transform some standard competent cells (DH5a or likewise), cultivate them, make a prep and check your plasmid at least on a gel, ideally but restriction digest. Also make some glycerol stocks. So you make sure you have the right plasmid and you have a stock you can always go back to. $\endgroup$
    – Chris
    Nov 15, 2023 at 11:30
  • $\begingroup$ I will consider this after my current experiment batch. I will do 5 plates, 2 with Ecoli 21D3 and two with NEB5 alpha, two different antibiotic concentration and 1 plate just with LB media at the smaller antibiotic concentration. Therefore I should be able to reduce the amount of possible error sources based on the outcome of the experiment. When not a single of 4 plates show colonies I agree, the integrity of the plasmid is in doubt. $\endgroup$
    – raptorlane
    Nov 15, 2023 at 12:16
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    $\begingroup$ I think that your title here is entirely misleading - you aren't looking at protein expression in this case, simply transformation of the bacteria with a plasmid. Frist step is to test that your system works with your cells and plasmid and with known good cells and plasmid - beg/borrow known good competent cells and plasmid from a colleague and test them in parallel with your cells and plasmid. Note that you need competent cells for this, not just bacterial culture. $\endgroup$
    – bob1
    Nov 15, 2023 at 20:30

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In my case it helped to treat the filterpaper longer with miliq water to increase plasmid concentration. I also used more plasmid in the 3rd attempt and it worked. Miniprep was performed nevertheless. When a similar experiment is not working, I would recommend to increase plasmid concentration, increase incubation time and decrease antibiotics in that order. I tested the plasmid on agarose gel and further I sent the plasmid further to a sequencing service for validation. Seems all to checkout.

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