Team:Heidelberg/Notebook/Killing I/Notebook/week11

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Revision as of 10:49, 29 October 2008

Week 11

Back to the overview

1 Monday, 10/13/08
- 1.1 Proceeding of new phage cloning strategy
- 1.2 Proceeding of cloning CmR and oriT in standard plasmid
2 Tuesday, 10/14/08
3 Wednesday, 10/15/08
- 3.1 new phage cloning strategy
4 Thursday, 10/16/08
- 4.1 new phage cloning strategy
5 Friday, 10/17/08
- 5.1 new phage cloning strategy
6 Saturday, 10/18/08
- 6.1 new phage cloning strategy
7 Sunday, 10/19/08
- 7.1 new phage cloning strategy

Monday, 10/13/08

Proceeding of new phage cloning strategy

Digestion of the miniprep 1-6,9,10 from sunday and the original pBluescript with insert

Digestion with XbaI/XhoI (top)
- normal: 4549, 2157, 34
- new: 3945, 2898

Digestion with SacI/SpeI (bottom)
- normal: 4549, 2157, 34
- new: 4549, 1331, 929, 34

Gel

- lane0: dna ladder mix
- lane1: sample 1
- lane2: sample 2
- lane3: sample 3
- lane4: sample 4
- lane5: sample 5
- lane6: sample 6
- lane9: sample 9
- lane10: sample 10
- lane11: pBluescript with insert

PCR with CmR_suffix and CmR_prefix (top)
- normal: 1668bp
- new: 852bp, 919bp

PCR with oriT_prefix and CmR_suffix (bottom)
- normal: 2150bp
- new: 1329bp

Gel

- lane0: dna ladder mix
- lane1: sample 1
- lane2: sample 2
- lane3: sample 3
- lane4: sample 4
- lane5: sample 5
- lane6: sample 6
- lane9: sample 9
- lane10: sample 10
- lane11: pBluescript with insert

--> we do not have a working GFP/CmR in pBlue/insert!!! --> do the ligation again (beginng from the mutagenesis pcr)

Proceeding of cloning CmR and oriT in standard plasmid

inoculation of CmR Std. Mutagenesis PCR sample and oriT Std. colonies

Tuesday, 10/14/08

Proceeding of cloning CmR and oriT in standard plasmid

Miniprep of 6 oriT and 5 CmR Std. samples
- digestion with EcoRI/PstI (to cut out insert of pSB1A2)

- lane0: dna ladder mix
- lane1-6: oriT 1-6
- lane7: 1kb dna ladder plus
- lane8: CmR Std. Mut 1.1.1
- lane9: CmR Std. Mut 1.1.2
- lane10: CmR Std. Mut 1.2.2
- lane11: CmR Std. Mut 2.2.1
- lane12: CmR Std. Mut 2.2.2

- expected fragments:
  - oriT: 2000bp, 500bp
  - CmR: 2000bp, ca. 900bp

-->no oriT is right

-->CmR 1.1.1, 1.1.2, 2.2.1, 2.2.2 look good
-->sequencing of 2.2.1 and 2.2.2
- sequencing results match perfect

Three ligations of oriT pcr product with pSB1A2 backbone (PstI, XbaI) (1:2.5, 2:5, 3:7.5 (µl, vector:insert))
- 40min at RT
- transformation, plated out on Amp plates

Screening PCR of oriT agar plates
- pcr with standard plasmid primers (VF2, VR) using Taq
- 12 pcr samples (1-6 only one colony, 7-12 three colonies)
- Gel

- lane0: dna ladder mix
- lane1-12: screening sample 1-12
- expected fragment length: ca. 500-600bp
-->sample 3 and 5 look good
-->inoculation of overnight cultures
-->sequencing of 3 and 5
- sequencing results match perfect

new phage cloning strategy

Mutagenesis PCR of pBlue with insert to remove KpnI restriction site
- using turbo Pfu
- elongation: 12,5min
- DpnI digestion 2h at 37°C
- transformation in TOP10, plated out on Cm plate

old phage cloning strategy

mutation of old insert in pBluescript
pBluescript + insert was cut with BamHI --> the resulting backbone cut out of the gel and religated. With this vector 2 mutagenesis PCRs were done to eleminate the two remaining XbaI restriction sites.
The resulting vector was digested with XbaI/XhoI to get the insert with the correct restriction sites for ligation into lambda phage

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 <body>
@@ Line 52: / Line 393: @@
 		</li>
 		<li>
-			<a href="https://2008.igem.org/Team:Heidelberg/Chemotaxis" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
 			<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
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@@ Line 62: / Line 403: @@
 		</li>
 		<li>
-			<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
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 		</li>
 		<li style="width: 160px">
-			<a href="https://2008.igem.org/Team:Heidelberg/Project/General_information" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
+			<a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
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@@ Line 106: / Line 447: @@
                                 <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"><span><span>Surveys</span></span></a></li>
                                 <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"><span><span>Open Day</span></span></a></li>
                                 <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>
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Team:Heidelberg/Notebook/Killing I/Notebook/week11

From 2008.igem.org

Revision as of 10:49, 29 October 2008

Contents

Monday, 10/13/08

Proceeding of new phage cloning strategy

Proceeding of cloning CmR and oriT in standard plasmid

Tuesday, 10/14/08

Proceeding of cloning CmR and oriT in standard plasmid

new phage cloning strategy

old phage cloning strategy

Wednesday, 10/15/08

new phage cloning strategy

Thursday, 10/16/08

new phage cloning strategy

Friday, 10/17/08

new phage cloning strategy

Saturday, 10/18/08

new phage cloning strategy

Sunday, 10/19/08

new phage cloning strategy