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==="S"LN set up=== * For a final volume of 2.5 uL to each tube: 0.1 uL of 5 U/uL Ampligase (final = 0.5 U/uL), 0.25 uL of 1 mM dNTPs, 0.25 uL of 10X Ampligase Buffer. I added 0.60 uL of LN mix to each first. * Added LN mix at 20 hour 56.4C incubation. * For PfuTurboCX Hotstart, I aliquoted 1.5 uL that was then incubated at 95C for 3 minutes. Then transferred 1.2 uL to the capture reaction, (tube - PT). * I will use 3 U of each polymerases: {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Polymerase''' | align="center" style="background:#f0f0f0;"|'''Concentration (U/ul)''' | align="center" style="background:#f0f0f0;"|'''3 U Vol (uL)''' | align="center" style="background:#f0f0f0;"|'''Water (uL)''' |- | Stoffel||10||0.3||1.6 |- | HemoKlentaq||5||0.6||1.3 |- | KlenTherm||10||0.3||1.6 |- | PfuTurboCX||2.5||1.2||0.7 |- | Truncated Taq||5||0.6||1.3 |- | No-Polymerase||0||0||1.9 |} * After adding polymerases, I added water last. * After 20 hours, denature DNA at 95C for 3 minutes. '''The polymerases and Ampligase do not get heat inactivated easily.''' * Add 1 uL of Exo I and 1 uL of ExoIII. Incubate at 37C for 2 hours. * After ExoI/III, I performed amplification with Phusion Flash and saw a big smear in all my lanes. I believe this is due to the ExoI/III reaction, so I repeated ExoI&III digestion with new tubes of enzymes & repeated amplification.
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