Bioruffo
Molecular Biology protocols, utilities and oddities
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If you want to quickly check transformed colonies for the presence of an insert of at least 400-500bp, you can use this simple protocol. It will not be as informative as a PCR in terms of insert size, but it will allow you to identify plasmids of different sizes and thus spot the "empty" ones.
The protocol is adapted from this one from the Han Lab which I had originally found on the internet, with some changes. I use a 2X cracking lysis buffer, that doesn't need to be heated to dissolve the agarose; the quantity of buffer is proportinally increased. I also do a quick centrifugation before loading to pellet the debris, and only load the supernatant.
 
Protocol:
 
1) Prepare a 1% agarose gel;
   while it is chilling,
 
2) Prepare the 2X cracking lysis buffer.
      Weigh in an Eppendorf tube:
            Sucrose (or kitchen sugar)       200 mg
      Then add:
            Water                                     860 ul
            NaOH 5M                                  40 ul
            SDS 10%                                  20 ul
      Mix to dissolve, and add a nothingful of bromphenol blue or Xylene cyanol.
      That's just to give it some color and make gel loading easier.
 
3) Label one eppie tube for each colony, and add 20 ul of dH2O
      (or 25-30ul if you plan to take aside an aliquot for colony PCR).
4) Add the colonies to the tubes and mix briefly.
      The wise choose at least one blue colony as "empty" control.
      If you want to perform colony PCR on the same colony, separate an aliquot now.
5) Add 20ul cracking lysis buffer to the colonies and mix.
      If you have plenty of tubes, consider putting the 20ul inside the caps (no need to change the tip every time).
      Then close all tubes and spin them to pour the buffer on the colony. Then mix briefly.
6) Centrifuge, 6000g, 5 minutes.
      This is not in the original protocol, but I strongly advice doing so. Loading a solution "dirty" with debris on the gell will make it float out of the well!
7) Now the agarose gel should be ready; load 25-30ul of the supernatant on an the gel, and run at standard conditions (you'll see the plasmid around the 2kb-4kb region).
 
Expected results:
 
You should see on the gel:
-A bacterial genomic DNA band, equal in all colonies
-A plasmid DNA band, of various sizes depending on the insert length
-A RNA smear, with possibly one or two more definite bands around 500bp.
 
You won't be able to tell clearly the size of the insert, unless you don't have a supercolied DNA standard. But you'll see whether an insert is present or not.