Magni:Protocols/Digestion

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(Digestion)
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= Digestion =
= Digestion =
To perform a double digest with the Roche enzymes, add to a 0.2-ml PCR tube (25 ul total volume):
To perform a double digest with the Roche enzymes, add to a 0.2-ml PCR tube (25 ul total volume):
-
**1000 ng of DNA or up to 20.5 ul of DNA (if performing the excision of an insert for a ligation, e.g. E-P, X-P, E-S) or
+
**1000 ng of DNA (if performing the excision of an insert for a ligation, e.g. E-P, X-P, E-S) or
-
**500-700 ng of DNA or up to 20.5 ul of DNA (if opening a plasmid E-X or S-P for a ligation) or
+
**500-700 ng of DNA (if opening a plasmid E-X or S-P for a ligation) or
-
**50-1000 ng of DNA (200 ng is ok for most cases) or up to 20.5 ul of DNA (if performing a screening)
+
**50-1000 ng of DNA - 200 ng is ok for most cases - (if performing a screening)
*Milli-Q water up to 20.5 ul
*Milli-Q water up to 20.5 ul
*2.5 ul of buffer H
*2.5 ul of buffer H
*1 ul of the first enzyme
*1 ul of the first enzyme
*1 ul of the second enzyme
*1 ul of the second enzyme
 +
 +
IF THE DNA HAS A LOW CONCENTRATION AND THE REQUIREMENTS ABOVE CANNOT BE MET, USE UP TO 20.5 ul OF DNA
Mix very well and incubate in the thermocycler (''Digestion'' program, reaction volume of 25 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.
Mix very well and incubate in the thermocycler (''Digestion'' program, reaction volume of 25 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.

Revision as of 05:45, 9 November 2013

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Digestion

To perform a double digest with the Roche enzymes, add to a 0.2-ml PCR tube (25 ul total volume):

    • 1000 ng of DNA (if performing the excision of an insert for a ligation, e.g. E-P, X-P, E-S) or
    • 500-700 ng of DNA (if opening a plasmid E-X or S-P for a ligation) or
    • 50-1000 ng of DNA - 200 ng is ok for most cases - (if performing a screening)
  • Milli-Q water up to 20.5 ul
  • 2.5 ul of buffer H
  • 1 ul of the first enzyme
  • 1 ul of the second enzyme

IF THE DNA HAS A LOW CONCENTRATION AND THE REQUIREMENTS ABOVE CANNOT BE MET, USE UP TO 20.5 ul OF DNA

Mix very well and incubate in the thermocycler (Digestion program, reaction volume of 25 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.

NOTES

  • If in a hurry, digestion time for ligation purpose can be decreased up to 30 min.
  • Sometimes, a triple digest may be necessary. To perform it, set up the following reaction (35 ul total volume) in a 0.2-ml PCR tube:
    • 500-700 ng of DNA or up to 28.5 ul of DNA (for ligation purpose) or
    • 50-700 ng of DNA (200 ng is ok for most cases) or up to 28.5 ul of DNA (if performing a screening)
  • Milli-Q water up to 28.5 ul
  • 3.5 ul of buffer H
  • 1 ul of the first enzyme
  • 1 ul of the second enzyme
  • 1 ul of the third enzyme
Mix very well and incubate in the thermocycler (Digestion program, reaction volume of 35 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.
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