User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/08

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Library preparation by Nextera DNA sample Prep kit

Gel extraction

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 1. Excision of the DNA fragments (500 - 800 bp)
 2. Put on a filter tip (1,000) in 1.5 ml tube
 3. Incubation at -20C for 20 min
 4. Centrifusion at 17,900 x g for 5 min
 5. Purification of the flowthrough by qiaquick PCR purification kit
 6. 50 ul of buffer EB -> 1.5 ng/ul


Addition of emPCR-Compatible Sites and Library Enrichment

Reaction mixture conc. '
Recovered DNA fragment library (12.5 ng start) ?? 2.5
Nextera PCR buffer 2x 12.5
Nextera Primer Cocktail 50x 0.5
Nextera Adaptor1 50x 0.5
Nextera PCR enzyme 2.5 U/ul 0.5
H2O 8.5
total 25
72C 3 min
95C 30 sec
95C 10 sec
55C 30 sec
72C 15 sec
18 cycle

load 3.0 ul of sample

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Gel extraction by QIAEX II Extraction Kit

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Excised gel weight = 180 mg

Eluted with buffer EB 20 ul x 2

Concentration; 1.5 ul