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===Blunting(polishing) and Repairing reactions (QUICK blunting kit, NEB)(DNA Pol.I, USB)(09-26-'10)=== *Samples arrangement: No.1 No.2 No.3 No.4 No.5 No.6 -------------------------------------------------------------------- S1 Exonuclease (2uL)(20U/uL,USB) 0m 0m 30m 30m 60m 60m DNA Pol.I(1uL)(10U/uL,USB) - yes - yes - yes --- adding 1uL *Set up blunting reactions on the cold block: 1rxn 6.5rxn ----------------------------- H2O 4.0 26.00 10X buf 2.5 16.25 1mM dNTP 2.5 16.25 Blunt EZ(NEB) 1.0 6.50 Sample 15.0 - ----------------------------- 25.0 65.0/6.5 =10 --- Add DNA sample 15uL *Briefly vortexing the blunting reaction: Incubate at RT for 30min *For reactions need to add DNA Pol.I, directly add it on the top regardless the changes of total volume. *Directly purify the Blunt reaction products using QIAquick without inactivation step. **Mix 5Vol. PB buffer(125uL) with Blunted products(25uL) and then follow the purification procedures. **Elute the purified DNA using 15uL prewarmed H2O. Nanodrop results: [[File:sam092610_nanodrop.jpg|800px]] *DNA recovery rate calculation: **Loading 500ng (2000ng/100x25)sheared DNA on each of wells for size selection. **Size select the 1kb regions and Zymo-column purified -> Quick blunt -> QIAquick purified -> Eluted in 15uL H2O. **Total recovered amount ~150ng. 150/500 = ~1/3 **'''About 1/3 of DNA recovered from loaded DNA in gel.'''. Given that I only cut a partial region (1/2~1/3) of sheared DNA for recovery, the recovery rate is actually very good.
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