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We inserted GFP-gene into plasmid Bluescript SK+, transformed E. coli with this construct, and then plated on an agar plate with Ampicillin and X-gal to do a blue/white screening. We got blue colonies and white colonies, and normally the white colonies are considered positive for the insert and the blue ones are negative for the insert. The problem is that the results we got are actually the opposite. If we look at the plate under UV-light, we see that ALL the blue colonies are fluorescent, which means they actually produce the GFP, and ALL the white ones are not!! How would you explain this?? Thank you

Edit: i thought that maybe the insert could be in frame with lacz', so we get fusion proteins. However this doesn't explain why we got white colonies which are not fluorescent

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    $\begingroup$ What enzymes did you use to perform the insertion? Also, what happens if you transform the original non-cut plasmid? $\endgroup$
    – March Ho
    Commented Apr 22, 2015 at 11:08
  • $\begingroup$ i forgot this information. We used kpnI for the insertion, which does blunt-end cuts. We also plated bacteria transformed woth the non-cut plasmids and we got some white colonies and only one blue colony.. but none of them is fluorescent $\endgroup$
    – Alice
    Commented Apr 22, 2015 at 11:26
  • $\begingroup$ KpnI is not a blunt restriction enzyme. Are you sure you didn't confuse it with another enzyme? Also, if your negative control is giving white colonies, then your plasmids are likely to be defective. $\endgroup$
    – March Ho
    Commented Apr 22, 2015 at 11:28
  • $\begingroup$ Oh yes pardon, kpnI makes cohesive cuts $\endgroup$
    – Alice
    Commented Apr 22, 2015 at 12:05
  • $\begingroup$ If the antibiotic was not working for whatever reason, you could get growth of un-transformed bacteria. $\endgroup$
    – canadianer
    Commented Apr 22, 2015 at 19:02

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Given the fact that the negative control uncut plasmids are producing white colonies, it seems that there have been significant mutations within the lacZ gene in the plasmid, causing the uncut plasmids to become white due to inactivation of the gene.

I would suggest purchasing a new batch of pBluescript, or if that is not possible for whatever reason, perform a miniprep on one of the blue colonies and sequence its multiple cloning site to verify that the MCS is present and not mutated before continuing. If you are not going to buy a new batch of plasmids, it would also be prudent to verify the plasmid's length using gel electrophoresis.

The most likely explanation is that the plasmids are mutated, and therefore you are getting bad results in the blue/white screen.

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