Magni:Protocols/Digestion: Difference between revisions
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* 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: | * 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 | **500-700 ng of DNA (for ligation purpose) or | ||
**50-700 ng of DNA | **50-700 ng of DNA - 200 ng is ok for most cases - (if performing a screening) | ||
*Milli-Q water up to 28.5 ul | *Milli-Q water up to 28.5 ul | ||
*3.5 ul of buffer H | *3.5 ul of buffer H | ||
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*1 ul of the second enzyme | *1 ul of the second enzyme | ||
*1 ul of the third enzyme | *1 ul of the third enzyme | ||
IF THE DNA HAS A LOW CONCENTRATION AND THE REQUIREMENTS ABOVE CANNOT BE MET, USE UP TO 28.5 ul OF DNA | |||
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. | 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 02:46, 9 November 2013
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 (for ligation purpose) or
- 50-700 ng of DNA - 200 ng is ok for most cases - (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
IF THE DNA HAS A LOW CONCENTRATION AND THE REQUIREMENTS ABOVE CANNOT BE MET, USE UP TO 28.5 ul OF DNA
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.
