imran.mungrue@utoronto.ca
*To remaining digestion (20ul) add (if necessary):
FOR BLUNT: T4 Pol 1ul (3U)
dNTP 2ul (10mM)
Incubate @12degC for 30min
Heat kill @ 75degC
FOR CIAP: CIAP 1ul
CIAP Buffer 2ul (10X)
Incubate @37degC for 15min
Heat kill @ 75degc
*Run sample on a 1% LMP gel with EtBr.
*Cut bands of interest out of gel with a razor, being sure too trim off excess gel that does not fluoresce with EtBr.
*Wash bands in 1ml water.
*Melt bands @ 65degC for 10-15min.
*Combine vector + insert in 2:8, 3:7 ratios and also control 2:0, 0:8, 3:0 and 0:7, store at 37degC until ready.
*To each tube add 10ul Ligation mix, mix and place on ice for 5 min.
Ligation mix:
0.4ul 100mM ATP
2 Ligase (Pharmacia, 6.2WU/ul)
2 10X OPA Buffer (Pharmacia)
5.6 water
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10
*Incubate O/N @ 15degC.
*Next day add 80ul water (or TCM) to each tube and melt @ 65degC.
TCM:
100ul Tris pH7.5 (1M)
100 MgCl2 (1M)
100 CaCl2 (1M)
9.7ml Water
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10ml
*Use 20ul for transformation in 50-60ul competent bacteria.