IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/25

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(Team Vector)
(Team Vector)
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Last week we had made cultures of V9, V10, V11 and V12 vectors and stored pellets of the resulting cells over the weekend.
Last week we had made cultures of V9, V10, V11 and V12 vectors and stored pellets of the resulting cells over the weekend.
-
We miniprepped the cells to obtain the plasmids. We did so using the Qiaquick Miniprep kit. As an initial test that we had obtained the plasmid we expected we digested using EcoI and HindIII and ran on a gel. The gel (shown below) showed the presence of the expected bands.
+
We miniprepped the cells to obtain the plasmids. We did so using the Qiaquick Miniprep kit. As an initial test that we had obtained the plasmid we expected we digested using PstI and HindIII and ran on a gel. The gel (shown below) showed the presence of the expected bands.
To verify that we had inserted the sequence that we expected into the plasmid we planned on sending them for sequencing by GeneWiz. GeneWiz require concentrations of 80ng/uL. Our nanodrop results showed that we had obtained low concentrations from our miniprep (<44ng/uL). To achieve higher concentrations of plasmid we re-picked colonies and incubated cultures for longer. We miniprepped the cultures and obtained higher concentrations of the plasmid.  
To verify that we had inserted the sequence that we expected into the plasmid we planned on sending them for sequencing by GeneWiz. GeneWiz require concentrations of 80ng/uL. Our nanodrop results showed that we had obtained low concentrations from our miniprep (<44ng/uL). To achieve higher concentrations of plasmid we re-picked colonies and incubated cultures for longer. We miniprepped the cultures and obtained higher concentrations of the plasmid.  
We did not have a large enough volume of plasmid for sequencing so we transformed the miniprepped plasmid back into E. coli and grew on LB + Kanamycin. We picked colonies and grew cultures. We obtained the plasmid using a miniprep.
We did not have a large enough volume of plasmid for sequencing so we transformed the miniprepped plasmid back into E. coli and grew on LB + Kanamycin. We picked colonies and grew cultures. We obtained the plasmid using a miniprep.
 +
 +
To create the insert for the V7 and V8 vectors we annealed oligos. We ligated the insert into the V7 and V8 backbones and transformed into E. coli. We incubated the E. coli overnight on LB + Kanamycin plates. Colonies grew on our negative control and colonies were unevenly distributed on other plates. This suggests that there may have been a problem with our plates or ligation so we should repeat those steps.
 +
 +
We did not have much O1 or O2 backbone remaining so we digested the V1 and V2 plasmids with SacII and SpeI to obtain more. We ran on a gel and gel purified the backbone.
==Team Flavor==
==Team Flavor==

Revision as of 14:02, 25 June 2010

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Week 2 Summary (6/25/2010)

Team Vector

Last week we had made cultures of V9, V10, V11 and V12 vectors and stored pellets of the resulting cells over the weekend.

We miniprepped the cells to obtain the plasmids. We did so using the Qiaquick Miniprep kit. As an initial test that we had obtained the plasmid we expected we digested using PstI and HindIII and ran on a gel. The gel (shown below) showed the presence of the expected bands.

To verify that we had inserted the sequence that we expected into the plasmid we planned on sending them for sequencing by GeneWiz. GeneWiz require concentrations of 80ng/uL. Our nanodrop results showed that we had obtained low concentrations from our miniprep (<44ng/uL). To achieve higher concentrations of plasmid we re-picked colonies and incubated cultures for longer. We miniprepped the cultures and obtained higher concentrations of the plasmid.

We did not have a large enough volume of plasmid for sequencing so we transformed the miniprepped plasmid back into E. coli and grew on LB + Kanamycin. We picked colonies and grew cultures. We obtained the plasmid using a miniprep.

To create the insert for the V7 and V8 vectors we annealed oligos. We ligated the insert into the V7 and V8 backbones and transformed into E. coli. We incubated the E. coli overnight on LB + Kanamycin plates. Colonies grew on our negative control and colonies were unevenly distributed on other plates. This suggests that there may have been a problem with our plates or ligation so we should repeat those steps.

We did not have much O1 or O2 backbone remaining so we digested the V1 and V2 plasmids with SacII and SpeI to obtain more. We ran on a gel and gel purified the backbone.

Team Flavor

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