Team:Goettingen/NoteBook w7
From 2013.igem.org
Line 4: | Line 4: | ||
</style> | </style> | ||
<div class="monat">July</div> | <div class="monat">July</div> | ||
+ | |||
+ | <div class="tlob" id="tl_0716"> | ||
+ | <span class="date">16th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <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> | ||
+ | <div class="timeline-cont"> | ||
+ | <div class="Section1" style="layout-grid:15.6pt"> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Transformation | ||
+ | from 15.7.13</span></b></p> | ||
+ | |||
+ | <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:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Amp-plates:<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">negative control | ||
+ | = negative/no clones</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">transformation | ||
+ | of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate</span></p> | ||
+ | |||
+ | <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:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Cm-plates<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">Negative control | ||
+ | = negative</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">Clones on all | ||
+ | other plates including w/o insert control (forgot gel extraction of pSB1C3 | ||
+ | vector </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family: | ||
+ | "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-char-type:symbol; | ||
+ | mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family: | ||
+ | Wingdings">à</span></span><span lang="EN-US"> terminator insert is still in | ||
+ | reaction mix; since we didn’t <span class="SpellE">dephosphorylate</span> 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…</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US"><o:p> </o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Colony | ||
+ | PCR for RBS-GFP-Terminator C12 re-<span class="SpellE">trafo</span> clones 1 to | ||
+ | 10 (RT-C1 to RT-C10)<o:p></o:p></span></b></p> | ||
+ | |||
+ | <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 "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">According | ||
+ | to protocol from 10.7.13<o:p></o:p></span></p> | ||
+ | |||
+ | <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 "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">16x | ||
+ | <span class="SpellE">MasterMix</span><o:p></o:p></span></p> | ||
+ | |||
+ | <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 "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">Clones | ||
+ | 1 – 10 picked, streaked out on <span class="SpellE">LB<sup>Amp</sup></span>-plate | ||
+ | and used for inoculation of 25 µl reactions (blue tubes)<o:p></o:p></span></p> | ||
+ | |||
+ | <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 "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">Controls: | ||
+ | 1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and | ||
+ | RBS-GFP-Terminator C12 (yellow tubes)<o:p></o:p></span></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<div class="tlob" id="tl_0715"> | <div class="tlob" id="tl_0715"> |
Revision as of 13:40, 17 September 2013
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
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)
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
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
![](https://static.igem.org/mediawiki/2013/6/60/Goe-15.07.13-RT.png)
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)
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 |