Team:SVCE CHENNAI/Restriction
Restriction
Requirements for each sample:
- Ice and bucket/container
- 1) 8-tube strip, or (3) 0.6ml thin-walled tubes
- BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
- dH2O
- NEB Buffer 2
- BSA
- Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
- Thermal cycler
Procedure:
- Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
- Add 2.5ul of NEBuffer 2.
- Add 0.5ul of BSA.
- Add 0.5ul of EcoRI.
- Add 0.5ul of PstI.
- There should be a total volume of 20ul. Mix well and spin down briefly.
- Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.
- Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
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