IGEM:Groningen/Notebook/iGEM 2011/2011/05/20: Difference between revisions
Joyce Mulder (talk | contribs) (Autocreate 2011/05/20 Entry for IGEM:Groningen/Notebook/iGEM_2011) |
Joyce Mulder (talk | contribs) |
||
(3 intermediate revisions by the same user not shown) | |||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==20-5-11== | ||
<br> | |||
<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 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. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 02:46, 1 August 2011
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
20-5-11
|