IGEM:Groningen/Notebook/iGEM 2011/2011/07/25: Difference between revisions
Joyce Mulder (talk | contribs) (Autocreate 2011/07/25 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. ##### --> | ||
== | ==25-7-11== | ||
<br> | |||
<br> Plates of transformants with PBAD-cI-LVA-DT and PBAD-RBS-GFP-DT vector look good, since the self ligation plates don't have much colonies! | |||
<br> 10μl plate is empty on self ligation plate control! 90μl plate has a few colonies... | |||
<br> Colony PCR as usual-> PBAD-cI-LVA-DT looks OK-> grow colony 8 and 9 overnight. | |||
<br> | |||
<br> Digestion PBAD, PhybB and vectors: cI-LVA and RBS-GFP | |||
<br> | |||
<br>PBAD: | |||
<br> 15μl PBAD | |||
<br> 1μl SpeI | |||
<br> 2μl Tango buffer | |||
<br> 2μ MQ water | |||
<br> | |||
<br>PhybB | |||
<br> 3μl PhybB | |||
<br> 1μl SpeI | |||
<br> 2μl Tango buffer | |||
<br> 14μl MQ water | |||
<br> | |||
<br> After 2h: put 2.5μl Tango buffer+ 1μl EcoRI in these samples and digest for another 2h. | |||
<br> DNA clean up with kit of Roche. | |||
<br> | |||
<br> Digestion vector (with LasR-LVA-DT): | |||
<br> | |||
<br> 3μl vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl Fast digest buffer | |||
<br> 1μl Fast AP | |||
<br> 21μl MQ water | |||
<br> | |||
<br> Ligation: | |||
<br> PBAD-LasR-LVA-DT | |||
<br> 6μl PBAD | |||
<br> 8.5μl cI-LVA-DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.5μl MQ water | |||
<br> | |||
<br> PhybB-LasR-LVA-DT | |||
<br> 6μl PhybB | |||
<br> 8.5μl cI-LVA-DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.5μl MQ water | |||
<br> | |||
<br> | |||
<br> Ligate for 30 to 45 minutes | |||
<br> | |||
<br> Transformation as usual | |||
<br> | |||
<br> Plasmid prep of LasR-LVA-DT colonies 6 and 7. DNA Concentration according to the N-D1000 Nanodrop is 46.7 and 51 ng/μl <br> respectively. | |||
<br> Colony 6 plasmid prep sample was used for vector sample today | |||
<br> Colonies 6 and 7 were send for sequencing and a glycerol stock has been made. | |||
<!-- ##### 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 07:22, 25 July 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> |
25-7-11
|