User:Tk/Notebook/MF/2009/11/24

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Mesoplasma florum simplification
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 * style="background-color: #F2F2F2" align="center"|  |Main Page


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16S PCR and random cloning of fragments

 * Dilute L1, GF, PP, Ent DNA to 10 ng/ul in TE


 * PCR reaction master mix
 * 100 &mu;l Qiagen Syber green m/m
 * 3 &mu;l each of primers for ends of RNA gene (1.5Kb)
 * 94 &mu;l water
 * aliquot 50 &mu;l add 1 &mu;l of diluted DNA
 * Cycle
 * 95 2:00
 * 95 :30
 * 55 :30
 * 68 1:40 X37

Cut pUC19 DNA with EcoRI
 * 50 &mu;l reaction with 1 &mu;g DNA
 * 30 min/heat kill

Phosphatase treat 1/2 of the reaction
 * Aliquot and add 2.5 &mu;l Antarctic phosphatase buffer to 25 &mu;l, add 1 &mu;l antarctic phosphatase
 * 30 min/heat kill

Ligate with EcoRI digests from 11/21/09
 * Master mixes of 1 &mu;l cut pUC19, 1 &mu;l T4 ligase, buffer
 * Add 2 &mu;l EcoRI cut DNA
 * ligate at RT 20 minutes, then 20 min at 4C

Transform onto XGal + IPTG plates (actually S-Gal)


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