Magni:Protocols/Digestion: Difference between revisions
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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 (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) 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 | ||
| Line 13: | Line 13: | ||
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. | ||
= 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 10:43, 2 February 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 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.
