Lidstrom:Building with DNA

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(Verify with Sequencing)
(Verify with Sequencing)
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==Verify with Sequencing==
==Verify with Sequencing==
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*See our [[Lidstrom:Sequencing_with_GeneWiz "Sequencing with GeneWiz"]] page.
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*See our [[Lidstrom:Sequencing_with_GeneWiz| "Sequencing with GeneWiz"]] page.

Revision as of 00:06, 8 March 2012

Back to Protocols

Contents

Obtain Parts

Perhaps you have them already. Perhaps you will make BioBrick or BglBrick parts via PCR.

  • Making parts by PCR:
    • oligo design:
      • use Phusion enzyme to amplify. If you are adding DNA to the ends with primers, make the annealing temperature of the homologous region have Tm approximately 55oC; you shorten your homologous region to as few as 10 bp to achieve this. Use the finnzyme Tm calculator. The sequence of DNA you add with primers can be essentially whatever you want. Don't pay the oligo synthesis company for additional purification of oligos, even up to 100 bp+; instead verify your constructs with sequencing. Despite the manufacturers' claims that ~60% of an 100 bp oligo is incorrect/prematurely terminated in synthesis, the first clone is usually correct.
    • purify products
      • column purify or gel purify the product to remove salts, oligos, etc.
        • column purify if you get only 1 band; gel purify if you get multiple bands or if your construction fails after simply column purifying. Remember, column purification has ~90% yield whereas gel purification has ~ 90% loss.

Digest with Restriction Enzymes

  • Use 500+ ng of each part (more if you intend to gel purify), 5 uL of 10X buffer, 0.5 uL of BSA (stabilizes enzymes) and 1uL of each restriction enzyme, and water to 50uL.
  • 37oC 1.5 hrs, 80oC 20 min (if heat inactivation works with your enzymes), then 4oC forever works in Janet's limited experience.
  • Decide if/how to purify:
    • Gel purification removes salts and restriction enzymes, which can increase success with subsequent ligation.
      • If you are cutting an insert out of a plasmid, you need to remove the piece of the plasmid you don't want to ligate. Gel purify in this case so you can select the band with the appropriate size.
      • If you are cutting off small fragments from a PCR product, you can usually column purify because the fragments you cut off are small enough that they don't bind to the column. Recall that column purification has ~90% yield whereas gel purification has ~90% loss. If you are only going to column purify (or skip purification all together) consider running a few uL on a gel to make sure you only have 1 band.
      • Betsy always gel purifies. Amanda usually does but forgot once and it worked. The Ginkgo manual doesn't recommend purification.

Ligate

  • add 1 uL of ligase (kit allows 0.1 to 1 uL) to a 20 uL reaction.
  • Incubate on counter (~2 hours) or at 16oC overnight.
  • You can try varying ratios of vector:insert. Stoichiometries of 1:1, 1:3, 1:6 are common.
    • The BioBricks/Ginkgo manual suggests only 15 minutes of incubation at 37oC; I have not tried this because I have not been in a rush and because it is very short compared to standard practice I hear about via word of mouth. -Janet 2/24/12
  • There is no need to purify ligated products or heat-inactivate ligase before transformation.

Transform

  • transform ~10 uL of the product. There is some left over in case you need to try again.

Screen with Colony PCR

Verify with Sequencing

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