Team:Goettingen/NoteBook w7

From 2013.igem.org

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<div class="monat">July</div>
<div class="monat">July</div>
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<div class="tlob" id="tl_0716">
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<span class="date">16th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<div class="cont">
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<p class="timeline-title">Transformation from 15.7.13,Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)</p>
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<div class="timeline-cont">
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<div class="Section1" style="layout-grid:15.6pt">
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<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Transformation
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from 15.7.13</span></b></p>
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<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:&quot;Calibri&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:
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Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Amp-plates:<o:p></o:p></span></b></p>
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<p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">negative control
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= negative/no clones</span></p>
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<p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">transformation
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of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate</span></p>
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<p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:&quot;Calibri&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:
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Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Cm-plates<o:p></o:p></span></b></p>
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<p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">Negative control
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= negative</span></p>
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<p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">Clones on all
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other plates including w/o insert control (forgot gel extraction of pSB1C3
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vector </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:
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&quot;Times New Roman&quot;;mso-hansi-font-family:&quot;Times New Roman&quot;;mso-char-type:symbol;
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mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:
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Wingdings">à</span></span><span lang="EN-US"> terminator insert is still in
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reaction mix; since we didn’t <span class="SpellE">dephosphorylate</span> the
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vector with AP, there will be false positives corresponding to plasmid part
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7!); smaller and larger clones on all plates; further incubation over day since
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they were too small to be picked in the morning…</span></p>
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<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US"><o:p>&nbsp;</o:p></span></b></p>
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<p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Colony
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PCR for RBS-GFP-Terminator C12 re-<span class="SpellE">trafo</span> clones 1 to
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10 (RT-C1 to RT-C10)<o:p></o:p></span></b></p>
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<p class="MsoListParagraphCxSpFirst" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">According
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to protocol from 10.7.13<o:p></o:p></span></p>
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<p class="MsoListParagraphCxSpMiddle" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">16x
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<span class="SpellE">MasterMix</span><o:p></o:p></span></p>
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<p class="MsoListParagraphCxSpMiddle" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">Clones
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1 – 10 picked, streaked out on <span class="SpellE">LB<sup>Amp</sup></span>-plate
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and used for inoculation of 25 µl reactions (blue tubes)<o:p></o:p></span></p>
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<p class="MsoListParagraphCxSpLast" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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</span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">Controls:
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1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and
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RBS-GFP-Terminator C12 (yellow tubes)<o:p></o:p></span></p>
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</div>
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<div class="fbutton">Fold ↑</div>
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</div>
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</div>
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</div>
<div class="tlob" id="tl_0715">
<div class="tlob" id="tl_0715">

Revision as of 13:40, 17 September 2013

July
16th

Transformation from 15.7.13,Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)

Transformation from 15.7.13

-          Amp-plates:

negative control = negative/no clones

transformation of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate

-          Cm-plates

Negative control = negative

Clones on all other plates including w/o insert control (forgot gel extraction of pSB1C3 vector à terminator insert is still in reaction mix; since we didn’t dephosphorylate the vector with AP, there will be false positives corresponding to plasmid part 7!); smaller and larger clones on all plates; further incubation over day since they were too small to be picked in the morning…

 

Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)

-          According to protocol from 10.7.13

-          16x MasterMix

-          Clones 1 – 10 picked, streaked out on LBAmp-plate and used for inoculation of 25 µl reactions (blue tubes)

-          Controls: 1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and RBS-GFP-Terminator C12 (yellow tubes)

Fold ↑
15th

Analysis of the RD samples from 12.7

Analysis of the RD samples from 12.7.

Test run of plasmids purified on 12.7.13, vector and inserts on agarose gel

  • part 7 plasmid, purified on 12.7.13
  • pSB1C3 vector from part 7, purified on 11.7.13
  • plasmid RBS-GFP-Terminator C12
  • plasmid RBS-GFP-Terminator C12 test restriction with EcoRI
  • inserts of DarR/riboswitches after 2nd digest with EcoRI
  • 1% agarose-1xTAE-gel
  • 3 µl 2 log ladder loaded on both sides of gel as a marker

Samples:

Component

Part 7 plasmid

(179.3 ng/µl)

pSB1C3

(2nd elution, 3.4 ng/µl)

plasmid RBS-GFP-Terminator C12

(200.8 ng/µl)

plasmid RBS-GFP-Terminator C12 R.D.

DarR purified PCR product (4 µl primer; reaction from 24.6.13; 15.2 ng/µl)

DarR insert

DNA

0.5 µl

4 µl

0.5 µl

5 µl

2 µl

3 µl

dH2O

3.5 µl

-

3.5 µl

-

2 µl

1 µl

5xLD

1 µl

1 µl

1 µl

-(sample in green FD buffer

1 µl

1 µl

Component

Riboswitch iGEM_40/41

Purified PCR product

(45.7 ng/µl)

Riboswitch iGEM_40/41

insert

Riboswitch iGEM_41/42

Purified PCR product

(36.8 ng/µl)

Riboswitch iGEM_41/42

insert

Riboswitch iGEM_40/43

Purified PCR product

(47.7 ng/µl)

Riboswitch iGEM_40/43

insert

Riboswitch iGEM_42/43

Purified PCR product

(35.7 ng/µl)

Riboswitch iGEM_42/43

insert

DNA

1 µl

3 µl

-

3 µl

1 µl

3 µl

-

3 µl

dH2O

3 µl

1 µl

4 µl

1 µl

3 µl

1 µl

4 µl

1 µl

5xLD

1 µl

1 µl

1 µl

1 µl

1 µl

1 µl

1 µl

1 µl

(for Riboswitch iGEM_41/42 and _42/43, there were too small amounts of PCR product left -> control was not possible…)

  • Run at 100 V
  • EtBr staining + destaining in water
  • UV detection

Purification of DarR and Riboswitch inserts

  • Qiagen PCR purification kit
  • 500 µl PB used for each reaction
  • 2x elution in pre-warmed HPLC water (25 µl)
  • NanoDrop concentration measurements (for some samples, the concentration was measured twice, since first values for 1st and 2nd elution were a little strange… -> approximate average concentration can be used for further calculations)

1st elution

Sample

Concentration (ng/µl)

Average concentration

A260/A280

A260/A230

Riboswitch iGEM_40/41 insert

7.5

1.74

1.01

Riboswitch iGEM_40/43 insert

5.5

1.18

1.09

Riboswitch iGEM_42/41 insert

2.3

3

1.89

0.86

4.6

1.38

0.76

Riboswitch iGEM_42/43 insert

3.0

3

2.18

0.76

3.1

1.09

0.90

DarR insert

1.7

2

3.34

0.71

2.4

1.06

0.62

2nd elution

Sample

Concentration (ng/µl)

Average concentration

A260/A280

A260/A230

Riboswitch iGEM_40/41 insert

5.2

1.30

0.56

Riboswitch iGEM_40/43 insert

1.5

0.99

0.36

Riboswitch iGEM_42/41 insert

2.6

3

1.38

0.52

3.6

1.18

0.62

Riboswitch iGEM_42/43 insert

3.9

4

1.14

0.63

4.2

1.07

0.61

DarR insert

2.9

1.04

0.52

Fold ↑

Retrieved from "http://2013.igem.org/Team:Goettingen/NoteBook_w7"