Team:Goettingen/NoteBook w7
From 2013.igem.org
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Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10), Colony PCR for DarR and Riboswitch biobrick transformation from 15.7.13
Gel
run: Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to
RT-C10)
- Addition of 5 µl 5xLD to the samples
- Loading of 5 µl of each sample on 1 % agarose gel
- Loading of 3 µl 2 log ladder as a marker
- Gel run at 100 V
- EtBr staining + destaining in water
- UV detection
Re-Trafo Clones 1, 4 – 7, 9 and 10 look promising: could contain RBS-GFP-Terminator construct in pSB1A3 backbone
è Re-Trafo Clone 2 has a strange additional band (from plasmid?)
è Re-Trafo Clone 8: no product
è Even for Plasmid of
RBS-GFP-Terminator C12, the additional band at the height of the terminator PCR
product is not seen à but for plasmid GFP this band could be visible… additionally strong
band of GFP-Terminator C1 PCR product runs higher than band of PCR product for
RBS-GFP-Terminator C12 à maybe I mixed up with the samples…? If so, all clones would contain
the GFP-Terminator plasmid. But this cannot be…
è Sequencing of Re-trafo
clones 1, 4 and 5??? Ask Katrin G. about her opinion of
the gel!
- Samples stored at -20°C in “yellow-tip-box rag”
Gel
run: Colony PCR for DarR and Riboswitch biobrick transformation from 15.7.13
- Addition of 5 µl 5xLD to the samples
- Loading of 5 µl of each sample on 1 % agarose gel
- Loading of 3 µl 2 log ladder as a marker
- Gel run at 200 V
- EtBr staining + destaining in water
- UV detection
Gel 1:
Gel 2:
è Only for clone 4 two weak bands at 2 – 3 kb are seen, but this can actually not correspond to any of the biobricks. Could be the plasmid
è All clones are apparently negative corresponding to re-ligated pSB1C3-terminator… Don’t forget gel ex next time…XD
- Samples stored at -20°C in “yellow-tip-box rag”
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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)
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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 |