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Restricted
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Riachalder (Talk | contribs) (New page: Restricting Plasmids (Double Restriction) From 2008.igem.org Jess enjoying pipetting buffer into her restriction mixture.We have conducted restrictions in varying concentrations and total...) |
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| - | + | We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out. | |
| - | + | ||
| - | + | ||
| + | Dilute | ||
| - | + | * 46μl MillQ H2O | |
| - | + | * 10μl 10 x buffer | |
| - | 46μl MillQ H2O | + | * 40μl plasmid sample |
| - | 10μl 10 x buffer | + | * 2μl enzyme 1 |
| - | 40μl plasmid sample | + | * 2μl enzyme 2 |
| - | 2μl enzyme 1 | + | |
| - | 2μl enzyme 2 | + | |
Total volume = 100μl | Total volume = 100μl | ||
Concentrated | Concentrated | ||
| - | 10μl MilliQ H2O | + | * 10μl MilliQ H2O |
| - | 3μl 10 X buffer | + | * 3μl 10 X buffer |
| - | 10μl plasmid sample | + | * 10μl plasmid sample |
| - | 1μl enzyme 1 | + | * 1μl enzyme 1 |
| - | 1μl enzyme 2 | + | * 1μl enzyme 2 |
Total volume = 30μl | Total volume = 30μl | ||
| - | + | Incubate solutions for 90 minutes in a 37°C water bath. | |
| - | If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes. | + | |
| - | + | If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes. | |
Revision as of 14:56, 16 September 2008
We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.
Dilute
- 46μl MillQ H2O
- 10μl 10 x buffer
- 40μl plasmid sample
- 2μl enzyme 1
- 2μl enzyme 2
Total volume = 100μl
Concentrated
- 10μl MilliQ H2O
- 3μl 10 X buffer
- 10μl plasmid sample
- 1μl enzyme 1
- 1μl enzyme 2
Total volume = 30μl
Incubate solutions for 90 minutes in a 37°C water bath.
If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.
