Knight:TOPO TA cloning

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(Notes)
(Notes)
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==Notes==
==Notes==
*[[Tom Knight]] claims you can cut the reaction volume in half and it will still work fine.
*[[Tom Knight]] claims you can cut the reaction volume in half and it will still work fine.
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*Use fresh PCR product for best results.  (The A overhang tends to get chewed back over time).
+
*Use fresh PCR product for best results.  (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
*Your primers should NOT have 5' phosphates.  They interfere with topoisomerase I.
*Your primers should NOT have 5' phosphates.  They interfere with topoisomerase I.

Revision as of 16:30, 29 September 2005

Notes

  • Tom Knight claims you can cut the reaction volume in half and it will still work fine.
  • Use fresh PCR product for best results. (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
  • Your primers should NOT have 5' phosphates. They interfere with topoisomerase I.

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