Team:Goettingen/NoteBook w7

From 2013.igem.org

18th

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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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)

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Concentration of dialysed protein samples
Determination of final protein concentration via Bradford test
SDS Page of dialysis and elution samples

Concentration of dialysed protein samples

-       Put E1 samples from dialysis in vivaspin column

-       Spin column for 20min at 4000xg

-       Add 4ml of E2 to vivaspin and centrifuge for 20min at 4000xg

-       Resuspend and repeat centrifugation and resuspension

 

 

Determination of final protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H2O) to 5µl of 1:20 diluted protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD595 / Vx0,0536

Eluate

Absorption (nm)

C [µg/ml]

Sample 1

0,121

0,49

Sample 2

0,123

1,49

Total: 9,35 mgml

 

SDS Page of dialysis and elution samples

-       Mix 15µl of samples  with 5µl Pab and 5µl buffer W

-       Incubation for 10min at 93°C

-       Mix each sample with 4µl protein marker

-       Load whole volume on 15% SDS-gel

-       Run for 5min at 90V and 1h at 120V

D1 | D2 | D3 | M | E1 | E2 |

 

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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


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