Lidstrom:Building with DNA

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Obtain Parts)
(Ligate)
Line 17: Line 17:
==Ligate==
==Ligate==
*add 1 uL of ligase (kit allows 0.1 to 1 uL) to a 50 uL rxn
*add 1 uL of ligase (kit allows 0.1 to 1 uL) to a 50 uL rxn
-
*Incubate on counter (~2 hours) and then in my fridge overnight or at 4oC overnight.
+
*Incubate on counter (~2 hours) or at 16oC overnight.  The BioBricks/Ginkgo manual suggests only 15 minutes of incubation at 37oC; I have not tried this because it is much shorter than the New England Biolabs suggested times.
==Transform==
==Transform==

Revision as of 20:24, 24 February 2012

Back to Protocols

Contents

Obtain Parts

  • Perhaps you have them already. Perhaps you will make BioBrick or BglBrick parts via PCR.
  • If you make parts via PCR, column purify the product to remove salts, oligos, etc.

Digest with Restriction Enzymes

  • Use 500+ ng of each part, 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.5

Ligate

  • add 1 uL of ligase (kit allows 0.1 to 1 uL) to a 50 uL rxn
  • Incubate on counter (~2 hours) or at 16oC overnight. The BioBricks/Ginkgo manual suggests only 15 minutes of incubation at 37oC; I have not tried this because it is much shorter than the New England Biolabs suggested times.

Transform

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

Screen with Colony PCR

  • See our colony PCR page for details.

Verify with Sequencing

  • See "Sequencing with GeneWiz" page.
Personal tools