My cloning attempt into a plasmid yielded colonies following transformation. Now what?

For Standard operating procedures

Congratulations!  But…before celebrating too much, make sure to check that your insert is what you think it is.  Here are things you should work on now:

  1. Pick a colony or two from the plate and spot some of the bacteria onto another plate (aka masterplate) and shake the rest of the bacteria off into 5mL LB broth.
    • Make sure the plate and broth is supplemented with the correct antibiotic.
  2. Incubate plate and liquid culture at 37C overnight.
    • The liquid culture should shake at ~220rpm
    • Store the masterplate at 4C
    • Purify the plasmid from the bacteria in the liquid culture using a plasmid miniprep kit
  3. Digest the vector with cut 800-1000ng of plasmid in a 10uL reaction containing your gene
    • Cut 800-1000ng of plasmid in a 10uL reaction
    • Use 0.5 ul of each restriction enzyme
    • Incubate for at least 2 hours at 37C (unless the recommended temperature for the enzyme is 25C!)
  4. Run the sample on at check that the insert is the correct size.  If your gene is cut into smaller pieces by either the restriction sites that flank your gene (for example: EcoRI and BamHI in pGBKT7), do the following:
    • Copy and paste the gene sequence into NEBcutter V2.0 and run the program
    • Look for the restriction enzyme you used to be listed in the 0 cutter list.  If it’s not there, look in the “1 cutter” and “2 cutter” lists.
    • Calculate the sizes of fragments you would expect from the restriction digests and compare these expectations with your gel image.
    • Note that small fragments of 200bp or less may be difficult/impossible to detect.
  5. If the fragment(s) is the correct length, make a glycerol stock
    • Pick bacteria from the appropriate colony on the masterplate
    • Inoculate and grow a 5mL culture as described above.
    • Pellet the bacteria and suspend in 400uL of bacterial freezing media (normal LB broth with 50% (v/v) glycerol)
    • Transfer to a well-labeled 1.5mL tube
    • Store in -80C glycerol stocks
    • Enter the information for this new construct into the log sheet for glycerol stocks (the sheet should be in a oligo information 3-ring binder)
  6. Sequence the entire length of the gene that is inserted into the vector.  We are set up to use the SimpleSeq DNA Sequencing Service from Eurofins.

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