Magni:Protocols/Digestion

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= Digestion =
= Digestion =
-
To perform a double digest with the Roche enzymes, add to a 0.2-ml PCR tube:
+
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 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
**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 or up to 20.5 ul of DNA (if opening a plasmid E-X or S-P for a ligation) or
-
**50-1000 ng of DNA or up to 20.5 ul of DNA (if performing a screening)
+
**50-1000 ng of DNA (200 ng is ok for most cases) or up to 20.5 ul of DNA (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
-
Mix well and incubate in the thermocycler (''Digestion'' program) for 2-4 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.
 +
 
 +
= 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.

Revision as of 13:43, 2 February 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 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
    • 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
    • 50-1000 ng of DNA (200 ng is ok for most cases) or up to 20.5 ul of DNA (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

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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