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Restricting Plasmids (Double Restriction)
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| + | {{:Team:Newcastle University/Template:UnderTheProtocol|page-title=[[Restricting Plasmids (Double Restriction)]]}} | ||
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| + | [[Image:Jess1.jpg|thumb|right|200px| Jess enjoying pipetting buffer into her restriction mixture.]] | ||
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. | 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. | ||
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* 46μl MillQ H2O | * 46μl MillQ H2O | ||
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* 2μl enzyme 2 | * 2μl enzyme 2 | ||
Total volume = 100μl | Total volume = 100μl | ||
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'''Concentrated''' | '''Concentrated''' | ||
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* 1μl enzyme 2 | * 1μl enzyme 2 | ||
Total volume = 30μl | Total volume = 30μl | ||
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| + | [[Image:DSC01597.jpg|200px]] | ||
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| + | The DNA purification kit we use to purify enzymatic reaction mixtures. | ||
* Incubate solutions for 90 minutes in a 37°C water bath. | * 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. | ||
Latest revision as of 16:01, 28 October 2008
Newcastle University
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Home >> Wet Lab >> Protocols >> Restricting Plasmids (Double Restriction)
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.
- 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
The DNA purification kit we use to purify enzymatic reaction mixtures.
- 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.
