Team:KULeuven/29 July 2008

From 2008.igem.org

(Difference between revisions)

Latest revision as of 13:09, 7 October 2008

dock/undock dropdown

<< return to notebook			return to homepage >>
< previous friday	← yesterday	tomorrow →	next monday >

Lab Work

Wet Lab

Picture of the gel with the digest of B0015, J23022 and J23032 with Xba I. The faint ladder shows that they were not properly cut.

Only one ligation succeeded ([http://partsregistry.org/Part:BBa_R0084 R0084]+[http://partsregistry.org/Part:BBa_B0032 B0032]). We think that the others failed because the vectors weren't properly cut. Therefore, we did another digest. This time we cut the vector with Xba I, and only when this worked, we cut it with EcoR I. We did this because Xba I has problems with cleaving close to the ends of DNA fragments. The problem is that Xba I doesn't seem to cut at all. Perhaps there's something wrong with the enzyme. We are trying to overcome this problem by allowing the restriction with Xba I to continue overnight.

Some more plasmids were isolated from the cells using the Qiagen Miniprep Kit. It concerns parts used in the filter and in the inverter ([http://partsregistry.org/Part:BBa_J23022 J23022], [http://partsregistry.org/Part:BBa_J23109 J23109], [http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_I712074 I712074], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_R0084 R0084], [http://partsregistry.org/Part:BBa_B0032 B0032] and [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_R0011 R0011], [http://partsregistry.org/Part:BBa_F1610 F1610]). We also digested some of these isolated plasmids. The ones that were cut with EcoR I and Spe I succeeded ([http://partsregistry.org/Part:BBa_J23109 J23109], [http://partsregistry.org/Part:BBa_I712074 I712074] and [http://partsregistry.org/Part:BBa_R0084 R0084]) and were cut out of the agarose gel and were purified. The ones that were cut with Xba I failed ([http://partsregistry.org/Part:BBa_J23022 J23022], [http://partsregistry.org/Part:BBa_J23032 J23032] and [http://partsregistry.org/Part:BBa_B0015 B0015]) - we didn't get the expected result with gel electrophoresis.

Dry Lab

General

GFP primers were finished and ordered. Antisense LuxI primers were written down and will soon be ordered. The primers have been given their place in the notebook section of the wiki.

As we noticed problems with [http://partsregistry.org/Part:BBa_M30109 M30109] - the "physical DNA" description of the part on the Registry proves nothing good and Melbourne 2007 reported problems with the part - we contacted the Melbourne 2008 team. They work with this part, and we hope that they can help us out.

We did some research about the origin of the EnvZ::KmR mutation in V1012 and found the paper of Mizuno and Mizushima. Based on the information in that paper, we will make primers to check whether the transduction has been a succes (or not).

Modeling

Mainly chillin' out, memory was finished and complete model is functional.

Wiki

Modeling section got taken care of some more, work on new dropdown menu proceeds...

Quote Of The Day

Sigrid: Het is te hopen dat de getransduceerde cellen een groeischeut krijgen.
Stefanie: Daar wacht ik nu al 22 jaar op!

@@ Line 1: / Line 1: @@
+{{:Team:KULeuven/Tools/Styling}}
+{{:Team:KULeuven/Tools/Header}}
 {{:Team:KULeuven/Tools/New_Day/Date_Retriever}}
@@ Line 4: / Line 7: @@
 === Wet Lab ===
-Only one ligation succeeded (R0084+B0032). We think that the others failed because the vectors weren't properly cut. Therefore, we will try another digest. This time we will cut the vector with ''Xba'' I, and only when this works, we will cut it with ''Eco''R I. We do this because ''Xba'' I has problems with cleaving close to the end of DNA fragments.
+[[image:gel-29-7.jpg|right|thumb|365px|Picture of the gel with the digest of B0015, J23022 and J23032 with ''Xba'' I. The faint ladder shows that they were not properly cut.]]
+Only one ligation succeeded ([http://partsregistry.org/Part:BBa_R0084 R0084]+[http://partsregistry.org/Part:BBa_B0032 B0032]). We think that the others failed because the vectors weren't properly cut. Therefore, we did another digest. This time we cut the vector with ''Xba'' I, and only when this worked, we cut it with ''Eco''R I. We did this because ''Xba'' I has problems with cleaving close to the ends of DNA fragments. The problem is that ''Xba'' I doesn't seem to cut at all. Perhaps there's something wrong with the enzyme. We are trying to overcome this problem by allowing the restriction with ''Xba'' I to continue overnight.
+Some more plasmids were isolated from the cells using the Qiagen Miniprep Kit. It concerns parts used in the filter and in the inverter ([http://partsregistry.org/Part:BBa_J23022 J23022], [http://partsregistry.org/Part:BBa_J23109 J23109], [http://partsregistry.org/Part:BBa_J23032 J23032], [http://partsregistry.org/Part:BBa_I712074 I712074], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_R0084 R0084], [http://partsregistry.org/Part:BBa_B0032 B0032] and  [http://partsregistry.org/Part:BBa_B0034 B0034], [http://partsregistry.org/Part:BBa_C0012 C0012], [http://partsregistry.org/Part:BBa_R0011 R0011], [http://partsregistry.org/Part:BBa_F1610 F1610]). We also digested some of these isolated plasmids. The ones that were cut with ''Eco''R I and ''Spe'' I succeeded ([http://partsregistry.org/Part:BBa_J23109 J23109], [http://partsregistry.org/Part:BBa_I712074 I712074] and [http://partsregistry.org/Part:BBa_R0084 R0084]) and were cut out of the agarose gel and were purified. The ones that were cut with ''Xba'' I failed ([http://partsregistry.org/Part:BBa_J23022 J23022], [http://partsregistry.org/Part:BBa_J23032 J23032] and [http://partsregistry.org/Part:BBa_B0015 B0015]) - we didn't get the expected result with gel electrophoresis.
 === Dry Lab ===
-== Modeling ==
+==== General ====
+GFP primers were finished and ordered. Antisense LuxI primers were written down and will soon be ordered. The primers have been given their place in the notebook section of the wiki.
+As we noticed problems with [http://partsregistry.org/Part:BBa_M30109 M30109] - the "physical DNA" description of the part on the Registry proves nothing good and Melbourne 2007 reported problems with the part - we contacted the Melbourne 2008 team. They work with this part, and we hope that they can help us out.
+We did some research about the origin of the EnvZ::KmR mutation in V1012 and found the paper of Mizuno and Mizushima. Based on the information in that paper, we will make primers to check whether the transduction has been a succes (or not).
+==== Modeling ====
 Mainly chillin' out, memory was finished and complete model is functional.
-== Wiki ==
+==== Wiki ====
 Modeling section got taken care of some more, work on new dropdown menu proceeds...
-== Remarks ==
+== Quote Of The Day ==
+Sigrid: Het is te hopen dat de getransduceerde cellen een groeischeut krijgen. <br> Stefanie: Daar wacht ik nu al 22 jaar op!