User:Tk/Notebook/MF/2009/11/24: Difference between revisions
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(Autocreate 2009/11/24 Entry for User:Tk/Notebook/MF) |
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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 μl Qiagen Syber green m/m | |||
** 3 μl each of primers for ends of RNA gene (1.5Kb) | |||
** 94 μl water | |||
* aliquot 50 μl add 1 μl of diluted DNA | |||
* Cycle | |||
** 95 2:00 | |||
** 95 :30 | |||
** 55 :30 | |||
** 68 1:40 X37 | |||
Cut pUC19 DNA with EcoRI | |||
* 50 μl reaction with 1 μg DNA | |||
** 30 min/heat kill | |||
Phosphatase treat 1/2 of the reaction | |||
* Aliquot and add 2.5 μl Antarctic phosphatase buffer to 25 μl, add 1 μl antarctic phosphatase | |||
** 30 min/heat kill | |||
Ligate with EcoRI digests from 11/21/09 | |||
* Master mixes of 1 μl cut pUC19, 1 μl T4 ligase, buffer | |||
* Add 2 μl EcoRI cut DNA | |||
* ligate at RT 20 minutes, then 20 min at 4C | |||
Transform onto XGal + IPTG plates (actually S-Gal) | |||
Latest revision as of 12:45, 24 November 2009
 Mesoplasma florum simplification
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16S PCR and random cloning of fragments
Cut pUC19 DNA with EcoRI
Phosphatase treat 1/2 of the reaction
Ligate with EcoRI digests from 11/21/09
Transform onto XGal + IPTG plates (actually S-Gal)
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