IGEM:Groningen/Notebook/iGEM 2011/2011/06/07: Difference between revisions
Joyce Mulder (talk | contribs) (Autocreate 2011/06/07 Entry for IGEM:Groningen/Notebook/iGEM_2011) |
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<br>PCR hyBB with taq DNA polymerase. Testing strain E.coli TG1 and DH5alpha. | |||
<br>for E.coli TG1: isolate DNA with magnetic beads of the 7th floor. | |||
<br>Do PCR with the isolated DNA (concentration is very low, one co worker on the 7th floor told me to use 10 to 12 microliter of the <br>DNA and then it should work! (at least it always worked for her)) | |||
<br> | |||
<br>Taq 10× buffer: 5μl | |||
<br>dNTPs: 1μl | |||
<br>MgCl2: 3μl | |||
<br>taq 5u/μl: 0.25μl | |||
<br>Forward primer 10mM: 1μl | |||
<br>Reverseprimer 10mM: 1μl | |||
<br>template: 1μl, but 12 μl for TG1 sample! | |||
<br>MQ: 38.75μl (but for sample TG1 use 26.75μl MQ) | |||
<br>PCR conditions: | |||
<br>denaturation: 10min | |||
<br>Cycle 35× | |||
<br>denaturtaion: 30s | |||
<br>annealing: 30s (gradient for 60, 65 and 70 degrees) | |||
<br>extension: 1min | |||
<br>Final extension: 10min | |||
<br>Store infinite at 4 degrees | |||
<br>Analyse them on a 1% agarosegel | |||
<br>PhybB has correct size! | |||
<br>Can use it for cloning, since taq polymerase makes 1 mistake per 1000bp according to Martijn, and PhybB plus prefix and suffix | |||
<br>is around 450bp. | |||
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