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Restricting Plasmids (Double Restriction)
From 2008.igem.org
Newcastle University
GOLD MEDAL WINNER 2008
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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.
