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Ligation
From 2008.igem.org
To ligate our 2.2kb ncl08 fragment from pUC57-ncl08 into our 2 vector plasmids (pJWV021 and pGFP-rrnB), we followed the protocol below. Prior to ligation we did not isolate the 2.2kb fragment from the other resulting digest product (a 2.7kb fragment) because we will select for cells with the correct insert once we have transformed and plated the cells.
- 2.5μl of 10 x ligase buffer
- 6μl vector plasmid cut with relevant enzyme(s)
- 15μl of digested plasmid which contains the fragment to be inserted, digested with the same enzyme(s) as the vector plasmid
- 1.5μl of T4 ligase
= 25μl total volume
Control:
- 2.5μl of 10 x ligase buffer
- 6μl of vector plasmid cut with relevant enzyme(s)
- 15μl of MilliQ H2O
- 1.5μl of T4 ligase
= 25μl total volume
Mix well with pipette and incubate for 1 hour in a 37°C water bath.
