Team:Goettingen/NoteBook w7

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<li>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)</li>
<li>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)</li>
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<strong>1st elution</strong>
<strong>1st elution</strong>
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Revision as of 14:14, 16 September 2013

July
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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