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. '
AGM 15.6 ng/ul 0.5
Nextera Reaction Buffer LMW 5x 2
Nextera Enzyme mix (Roche Titanium-compatible) ?? 0.5
H2O 7
total 10
55 C 7.5 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 ?? 2.5 2.5
Nextera PCR buffer 2x 12.5 12.5
Nextera Primer Cocktail 50x 0.5 0.5
Nextera Adaptor1 50x 0.5 0.5
Nextera PCR enzyme 2.5 U/ul 0.5 0.5
H2O 8.5 8.5
total 25 25
72C 3 min 72C 3 min
95C 30 sec 95C 30 sec
95C 10 sec 95C 10 sec
55C 30 sec 55C 30 sec
72C 15 sec 72C 15 sec
21 cycle 23 cycle

load 3.0 ul of sample

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