Lidstrom:Building with DNA

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***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 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.  
***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.  
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*** Betsy always gel purifies. Amanda usually does but forgot once and it worked.5
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*** Betsy always gel purifies. Amanda usually does but forgot once and it worked. The [http://ginkgobioworks.com/support/ Ginkgo manual] doesn't recommend purification.
==Ligate==
==Ligate==

Revision as of 16:34, 6 March 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. 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

  • See our colony PCR page for details.

Verify with Sequencing

  • See "Sequencing with GeneWiz" page.
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