User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/09

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Yeast Culture Prep for Long Term Freezer Storage

From Revived Yeast Culture NCYC1001

1) Overnight culture on agar plate

Streaked out 50.0ul of growing culture onto a glucose+yeast extract plate

S. pombe - failed to grow - redo S. cerevisiae - good lawn

2) Prepare freezing media = 20% gycerol in sterile YM media. Aliquot into small freezer tube (1000ul).

3) Collect lawn with sterile toothpick and swirl in freezing media

4) Slow freeze in -30C

Library preparation by Nextera DNA sample Prep kit

Tagmentation Reaction

Reaction mixture conc. '
AGM15.6 ng/ul0.5
Nextera Reaction Buffer LMW5x 2
Nextera Enzyme mix (Roche Titanium-compatible)??0.5
55 C7.5 min
Purified by MinElute kit (qiagen)11 ul of buffer EB

Addition of emPCR-Compatible Sites and Library Enrichment

Reaction mixture conc. 1. 12.5ng start 2. 0.78ng start '
Recovered DNA fragment library??2.52.5
Nextera PCR buffer2x12.512.5
Nextera Primer Cocktail50x0.50.5
Nextera Adaptor150x0.50.5
Nextera PCR enzyme2.5 U/ul0.50.5
72C3 min72C3 min
95C30 sec95C30 sec
95C10 sec95C10 sec
55C30 sec55C30 sec
72C15 sec72C15 sec
21 cycle23 cycle

load 3.0 ul of sample


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