Team:Newcastle/Restriction digests
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+ | =Restriction digestion= | ||
- | + | ==Materials required== | |
+ | * Eppendorf tubes | ||
+ | * Pipettes | ||
+ | * Appropriate DNA/plasmid | ||
+ | * Appropriate restriction enzymes | ||
+ | * 10X buffer | ||
+ | * Water | ||
- | == | + | ==Procedures== |
- | + | Add the following as mentioned below to make up to a final volume of 20 µl of reaction mix: | |
- | + | # 15 µl of DNA/plasmid | |
- | + | # 1 µl of restriction enzyme 1 | |
- | + | # 1 µl of restriction enzyme 2 | |
+ | # 2 µl of 10X buffer | ||
+ | # 1 µl of water | ||
+ | Incubate the digestion mixture at 37°C for 3 hours | ||
- | + | ==Notes== | |
- | *No more than 10% of enzyme - | + | *No more than 10% of enzyme should be used for a single reaction - glycerol inhibits reaction |
- | *10x buffer must be diluted to 1x i.e. 10% final volume | + | *10x buffer must be diluted to 1x i.e. 10% final volume. |
- | + | ||
- | + | '''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' | |
- | + | ||
+ | {{Team:Newcastle/footer}} |
Latest revision as of 15:54, 5 August 2010
|
Contents |
Restriction digestion
Materials required
- Eppendorf tubes
- Pipettes
- Appropriate DNA/plasmid
- Appropriate restriction enzymes
- 10X buffer
- Water
Procedures
Add the following as mentioned below to make up to a final volume of 20 µl of reaction mix:
- 15 µl of DNA/plasmid
- 1 µl of restriction enzyme 1
- 1 µl of restriction enzyme 2
- 2 µl of 10X buffer
- 1 µl of water
Incubate the digestion mixture at 37°C for 3 hours
Notes
- No more than 10% of enzyme should be used for a single reaction - glycerol inhibits reaction
- 10x buffer must be diluted to 1x i.e. 10% final volume.
Go back to our Protocol List