One of our customers was wondering if drying E. coli on Nylon transfer membranes prior to denaturation effects the colony hybridization.
Further, they were wondering if after drying the membranes, would treating with SDS before denaturation effect the hybridization? The
answer is...NO, this will work! The hybridization will not be adversely affected. This is the procedure they used.
1. performed colony lifts
2. air dried nylon membranes with E. coli
3. incubated 3 minutes on 3MM paper saturated with 10% SDS.
4. incubated 5 minutes on 3MM with 0.5M NaOH, 1.5 M NaCI.
5. incubated 5 minutes on 3MM with 1.5 M NaCI, 0.5M Tris pH 7.5.
6. incubated 5 minutes on 3MM with 2X SSC.
7. UV crosslinked without drying.
8. prehybridized and hybridized as usual.