Team:Goettingen/NoteBook w6

From 2013.igem.org

July
12th

Mini Plasmid Preparation, Test Digest of Clone 12 RBS-GFP-Terminator (see gel 11.07.) with EcoRI

Mini Plasmid Preparation

-          3x 4 ml ON culture of plasmid part 7 clone 1

-          1x 4 ml ON culture of RBS-GFP-Terminator clone 12

-          With Macherey-Nagel kit

-          Elution in 30 µl HPLC water

-          Pooling of 3 samples of part 7

-          NanoDrop concentration measurement:

 

 

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

Part 7 plasmid

179.3

1.79

1.38

Plasmid

RBS-GFP-Terminator C12

200.8

1.87

1.83

 

è Samples stored at -20 °C (pink rag)

Test Digest of Clone 12 RBS-GFP-Terminator (see gel 11.07.) with EcoRI

 

Test Digest pipette as follows:

 

1,5µl DNA (~300ng)

0.1µl EcoRI FD

1.5µl FD Buffer

11µl H20

 

àincubation at 37°C for 45 minutes

àAnalysis on 1% gel

 

Gel:

Marker|RBS-GFP-Terminator Clone12

am geldoc 2013-07-12 17hr 17min

 

The test RD shows only one band indicating a successful cloning procedure. To be sure, the DNA will be used for another transformation hopefully leading to clean clones.

-          the plasmid DNA (RBS-GFP-TermC12) was analyzed on the 1%gel below.

2nd round of RD of DarR and Ribiswitches A-D with new protocol

after Katrin Gunkas Überprotokoll

 

àFirst, the RDs of yesterday were analyzed on a 1% gel for complete digestion.

 

Gel:

Marker|DarR|Riboswitch 40/41|40/43|42/43|42/41|-|RBS-GFP-TermC12|-|part7after2ndRound2ndElution

am geldoc 2013-07-12 15hr 48min

 

The Riboswitch constructs shoe strong signals, DarR seems to be very sparsely left over.

 

àThe RDs were purified using the PCR purification kit (500µl PB and elution in 25µl pre-warmed water)

àThe purified DNA was analyzed with nanodrop

 

nanodrop:

 

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

DarR biobrick

3.4

1.13

0.56

Riboswitch 40/41

9.1

1.63

1.08

Riboswitch 40/43

8.4

1.81

1.17

Riboswitch 42/41

5.7

2.26

1.00

Riboswitch 42/43

4.0

2.16

0.92

 

àOf the purified DNA, all 24µl left were used for the 2nd round of RD with EcoRI FD

The 5 Reactions were pipetted like this

 

24µl DNA

16µl Mastermix:

4µl FD buffer

4µl EcoRI FD

8µl H2O

 

àIncubation at 37°C for 45 minutes

àFrozen away at -20°C

Fold ↑

11th

Plasmid mini-prep, Enyzme test: ThermoScientific vs. NEB (Biobrick Assembly Kit) ,Further RD of yesterday´s new protocol with part 7,

Miniprep of frozen plasmids from part7 and part8 in order to have DNA for cloning

NanoDrop:

Sample

ng/µl

A260nm/A280nm

A260nm/A230nm

Part7 mini

120.5

1.83

1.99

part8 mini

113.9

1.86

2.11

 

Incubation of Colony PCR master plates

è Plates taken out and put ot 4 °C (luckily not overgrown)

Enyzme test: ThermoScientific vs. NEB (Biobrick Assembly Kit)

è Since PstI digest was incomplete, it might be that the enzymes don’t work properly

è Test all enzymes in their preferred buffers

Test plasmid: plasmid part 1 purified on 1.7.13 à Linearization in single digest with EcoRI(-HF), XbaI, SpeI or PstI

-          Reaction according to test restriction digest to analyze clones after transformation

Component

Volume

Plasmid part 1 (2 ng/µl)

1 µl

Enzyme

1 µl

10x buffer

1 µl

dH2O

7 µl

Total Volume

10 µl

è Preparation of Mastermix:

Component

Volume

Plasmid part 1

15 µl

dH2O

105 µl

è Preparation of enzyme and buffer in Epi tubes

Company

Enzyme

Buffer

Activity

ThermoScientific

EcoRI

EcoRI buffer

100 %; recommended buffer

EcoRI

Buffer 0

100 %

XbaI

Tango

100 %

SpeI

Tango

100 %

PstI

Buffer 0

100 %

NEB (Biobrick Assembly Kit)

EcoRI-HF

CutSmart

100 %; recommended buffer

EcoRI-HF

NEB2.1

100 %

XbaI

CutSmart

100 %

XbaI

NEB2.1

100 %; recommended buffer

SpeI

CutSmart

100 %

SpeI

NEB2.1

100 %; recommended buffer

PstI

NEB3.1

100 %; recommended buffer

PstI

NEB2.1

75 %

è then addition of 8 µl master mix

-          incubation at 37 °C for 2 h

-          gel run:

o   1 % agarose 1xTAE gel

o   3 µl 2 log ladder as a marker

o   Addition of 2 µl 5x LD to each reaction, then loading of 8 µl

o   Run at 100 V

o   EtBr staining, destaining in water

o   UV detection

 

è All digestions are partial; reaction of PstI in NEB2.1 is less complete than other digests

è Does not fit with complete digest of part 7 with EcoRI-HF in CutSmart buffer from 10.7.13; in this digest and in enzyme test different heating blocks were used à could they be the problem?

Yesterday´s Colony PCR of all constructs so far on 1% gel:

 

Gel:

Marker|Part8-plasmidDNA|GFP-Term-plasmid-DNA|Part8-w/o-insert-control|RBS-GFP-Term-C6|-7|-8|-9|-10|-11|-12|-13|-14|-15|-16|M

Marker|Part8-plasmidDNA|GFP-Term-plasmid-DNA|Part8-w/o-insert-control|RBS-GFP-Term-C17|-18|-19|part7-plasmidDNA|part7-w/o-insert control-C1|-2|-3|DarR-biobrick-C1|-2|RiboswitchC-biobrick|RiboswitchD-biobrick|M

è Clone 12 from RBS-GFP-Terminator transformation could be positive, at least partially

è Inoculation of clone 12 from RBS-GFP-Terminator transformation over night + MiniPrep

è Re-transformation of plasmid, since clone seems to bee impure (band for RBS-GFP-Terminator and RBS alone…)

Clones from RiboC and RiboD and DarR transformation are negative

 

Further RD of yesterday´s new protocol with part 7

Yesterday´s samples were analyzed with the nanodrop:

NanoDrop measurements from R.D. from yesterday:

Sample

ng/µl

A260nm/A280nm

A260nm/A230nm

purified part7 R.D. PstI

41.4

1.74

2.07

purified part7 2nd elute PstI

8.0

1.49

1.27

purified part7 R.D. EcoRI

4.2

1.35

0.77

purified part7 2nd elute EcoRI

2.0

0.84

0.51

Only the first elution of the RD with PstI looks promising. This sample was used for the further, second digest. For time reasons, the EcoRI digest was started in parallel to the 1% gel, that is supposed to confirm the complete digestion of part 7 by PstI. The following picture shows the progress of the digest at 30 additional minutes of RD (see yesterday).

Gel:

Marker|PstI RD Part 7

After 30 more minutes of digestion with additional enzyme

The digestion has not been completed, an additional step has to be added in the end.

2nd round of digest:

àpipetted one reaction (40µl) after Katrin Gunkas Über-Protokoll (see yesterday) using EcoRI FD by Thermo Scientific and the FD buffer:

20µl DNA (all that was left)

4µl FD Buffer

4µl EcoRI FD

12µl HPLC H2O

40µl reaction volume

àincubation at 37°C for 30 minutes

àsample put on ice and 3µl loaded on 1% gel.

àgel melted, samples lost

àconsidering the previous experience, the digest was continued with EcoRI. Additionally, to proceed the unfinished digest with PstI, that digest was added to the reaction.

The following things were added to the reaction:

2µl FD Buffer

3µl EcoRI FD

3µl PstI FD

12µl HPLC H2O

 

20µl leading to a 60 µl reaction (minus 3µl on the gel)

àincubation at 37°C for 45 minutes

à3µl loaded on 1% gel (forgot to, loaded 6µl with PB of purification kitàdidn’t run)

Gel:

Needs to be redone, I suggest with the second elution, so we don’t lose too much DNA.

àpurification of the sample using the PCR purification kit, 500µl (different from protocol) PB buffer, 2x elution with 25µl pre-warmed HPLC water

nanodrop

Sample

ng/µl

A260nm/A280nm

A260nm/A230nm

purified part7 R.D. after 2nd round

27

1.71

1.59

2nd elution

3.4

2.71

1.06

àstorage at -20°C, “white sticker Ute-Box”

Inoculation of 4ml cultures for miniprep tomorrow

Three cultures of part 7 cryo-stocks from 11.6. and one culture of the RBS-GFP-Terminator construct Clone 12, which seems to be carrying at least partly the right construct (see today´s gel). Incubated in the Brutraum

New 1st round Single digestion of DarR and Ribiswitches A-D with new protocol

after Katrin Gunka Überprotokol

9 µl PCR product

4 µl 10X Buffer F.D.

4 µl PstI F.D.

23µl dH2O

incubated for 30 min at 37°C

Gel:

Wells as follows: DarR, Riboswitches 40/41, 40/43, 42/41, 42/43

rest of restriction stored in -20 freezer (for further digestion more enzyme has to be added)

..But where is DarR?...

Fold ↑

Making 8 liters of expression culture of L. monocytogenes carrying gene for the DAC-domain for crystallization

Making 8 liters of expression culture of L. monocytogenes carrying gene for the DAC-domain for crystallization

-       50ml LB medium containing Ampicillin inoculated with cryo 1013 stock

-       incubation o/n at 37°C with agitation

-       measure OD600

-       inoculation of 1l LB medium containing Ampicillin to OD600=0,1

-       incubation for 2 hours at 37°C to an OD600=0,5-0,9

-       induction of expression by adding 1ml of 1M IPTG

-       incubation for 12-14h at 16°C with agitation

-       measure OD600

-       divide 1l of culture in twice 500ml

-       centrifugation at 5000 rpm for 10min at 4°C

-       resuspend the pellets in ice cold 20ml buffer W and take it in a falcon tube

-       pool both separated treated samples together

-       centrifugation at 5000 rpm for 15min at 4°C

-       discard supernatant

-       store pellet at -20°C

 

Protein purification

-       resuspend cells from one falcon (pellet from 40ml of expression culture) in 10ml buffer W

-       apply solution from each falcon twice to french press

-       centrifugation for 15min at 8500rpm at 4°C

-       transfer supernatant into ultracentrifugation tubes

-       Ultracentrifugation for 1h at 35000rpm at 4°C

-       Preparation of columns: add 1ml of 50% Strep-Tactin and equilibrate with 10ml buffer W

-       Divide supernatant received from ultracentrifugation into 2 equal volumes and fill it up to 30ml with buffer W

-       Load protein solution on prepared columns

-       Wash 5 times with each 2,5ml buffer W

-       Elute with 0,5ml (for eluate 1) or 1ml (eluate 2-4) of buffer E

 

Determination of protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H2O) to 3µl of protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD595 / Vx0,0536

 

Eluate

Absorption (nm)

C [µg/ml]

1 E1

0,078

0,49

2 E1

0,239

1,49

3 E1

0,092

0,57

4 E1

0,042

0,26

5 E1

0,075

0,47

6 E1

0,049

0,3

7 E1

0,053

0,33

8 E1

0,128

0,70

Total: 4,197 mg in 4ml

1 E2

0,143

2,57

2 E2

0,114

0,71

3 E2

0,169

1,05

4 E2

0,127

0,79

5 E2

0,061

0,38

6 E2

0,188

1,17

7 E2

0,121

0,75

8 E2

0,141

0,88

Total: 8,3 mg in 8ml

 

Dialysis of protein samples

-       Pool all samples from E1 (4ml)

-       Pool all samples from E2 (8ml)

-       Pool all samples from E3 (8ml)

-       Transfer eluates into dialysis bags

-       Put dialysis bags into 4500ml H2O + 500ml 10x dialysis buffer

-       Dialysis o/n with agitation

 

Fold ↑

10th

Transformation from 8.7.13, colony PCR, Restriction Digest of Part 7 with new Protocol

Transformation from 8.7.13

-          No clones on negative control

-          No clones for Ribo A and B biobricks

-          3 clones for w/o insert control à partial digest of part 7; on the gel, a band of the uncut plasmid runs at approx. the same height as the band for pSB1C3 backbone à uncut plasmid was apparently extracted from the gel, as well…

-          2 clones for DarR biobrick

-          1 clone for Ribo C biobrick

-          1 clone for Ribo D biobrick

Partial digests, low number of positive clones, false positives…:

Biobrick Assembly Kit Cloning strategy = SHIT

è We’ll proceed according to old school, i.e. protocol “Katrin Gunka”…

 

Colony PCR

è To screen more efficiently for clones of RBS-GFP-Terminator cloning

è To screen transformation from 8.7.13 for positive clones

-          For RBS-GFP-Terminator clones 6 – 19 and 1 clone from w/o insert ligation (part 8); additional controls: plasmid part 8; plasmid GFP-Terminator C1

-          For single clone from cloning of Ribo D and Ribo C, both clones for DarR biobrick cloning and for all 3 clones from w/o insert ligation (part 7); additional control: plasmid part 7

-          With primers VF2 and VR

-          Prepare MasterMix (here: for 30 reactions)

Component

1x reaction

30x reaction

Taq buffer 10x

2.5 µl

75 µl

Taq Pol

1 µl

30 µl

dNTP mix (12.5 mM each)

1 µl

30 µl

Primer fwd (VF2)

1 µl

30 µl

Primer rev (VR)

1 µl

30 µl

dH2O

18.5 µl

555 µl

Total

25 µl

750 µl

è Distribute 25 µl of the master mix into the tubes

è For plasmid controls: preparation of 1:10 dilution of each plasmid in dH2O and addition of 1 µl of dilution to a tube; then addition of 25 µl master mix

-          pick clones, streak out on a master plate with a toothpick, then use same toothpick to inoculate PCR reaction

-          PCR protocol (30 cycles)

Step

Temperature

Time

Initial denaturation

94 °C

5 min

Denaturation

94 °C

45 sec

Annealing

54 °C

40 sec

Elongation

72 °C

4 min

Final Elongation

72 °C

10 min

Hold

15 °C

-          Incubation of master plate over day at 37 °C

Products stored at -20°C in a pipette-tip-stand

 

Restriction Digest of Part 7 with new Protocol

We now changed the protocol to single digest steps with purification instead of heat-inactivation.

Two digests were prepared, one with PstI and one with EcoRI-HF. For the double digest to be achieved, in a second digest, the products will be digested with the other enzyme, EcoRI-HF or PstI, respectively. This way, we try to find out, whether one enzyme inhibits the other enzyme´s function through its restriction. In the future, we will only need one of the two reactions, either starting PstI, or the other way around.

1st round of digest:

Two reactions were pipetted after Katrin Gunkas “Über-Protocol” as follows:

1: PstI digest

2: EcoRI-HF digest

 

1,5µg DNA (13µl for the isolated Part 7 DNA at 115.6ng/µl)

4µl Buffer (1: 3.1 for PstI / 2: CutSmart for EcoRI-HF)

4µl Enzyme (1: PstI by NEB / 2: EcoRI-HF by NEB)

19µl HPLC H2O

40µl reaction volume

 

àincubation at 37°C for 2h

à3µl on 1% gel to confirm complete digestion, undigested part 7 DNA as control (ATTENTION: Part 7 DNA almost empty)

 

Gel:

marker | uncut Part 7 | PstI RD | EcoRI RD

am geldoc 2013-07-10 16hr 48min

The EcoRI seems to be done cutting (although signal very low. Discussion: Since Part 7 was almost empty, I had to shake down the tube. This way the used DNA might have been diluted by condensed water, which I shook down). The PstI sample was put back onto the heat block at 37°C for 30 minutes with 3µl Enzyme added.

àpurification of the samples using the PCR purification kit, 500µl (different from protocol) PB buffer, 2x elution with 25µl pre-warmed HPLC water

àstorage at -20°C, “white sticker Ute-Box”

 

ànanodrop measurements have to be done tomorrow

è This R.D. was done using the NEB enzymes from the distribution kits. And all our cloning reactions using PstI did not work! In contrast, the test restriction digests that were done with the Fermentas enzymes EcoRI and PstI worked. Apparently, PstI enzyme from the distribution kit does not cut!

è Test PstI from distribution kit vs. Fermentas enzymes.

If this is true, then we have to prepare part 8 vector (SpeI, PstI cleavage) and GFP-Terminator cassette (XbaI, PstI cleavage) and all our inserts for RiboA, B, C, D and DarR biobrick again! Luckily, the PCR products and the plasmids are all at – 20 °C. So just another R.D. and another gel ex/ PCR clean-up…^^

Fold ↑

09th

DarRoperator PCR: 4 % gel run to check if PCR worked, preparation of antibiotics, test restriction.

Preparation of plates from 500 ml LBCm plates (35 µg/ml Cm)

Further incubation of RBS-GFP-Terminator back-up plate

è Taken out in the evening (clones appear not green)

Cryostock of GFP-Term C1 overnight culture

stored at -80

Mini-prep of

Part7 C1

GFP-Term C1

RBS-GFP-Term C1

RBS-GFP-Term C2

RBS-GFP-Term C3

RBS-GFP-Term C4

RBS-GFP-Term C5

eluted in 30 µl HPLC

Nano Drop:

Sample

ng/ µl

260/280

260/230

GFP-Term gel ex

6.8

1.84

0.10

pSB1C3

15.6

1.72

0.15

part 7 C1

115.3

1.91

2.22

GFP-Term C1 mini

111.0

1.90

2.19

RBS-GFP-Term C1

285.7

1.89

2.26

RBS-GFP-Term C2

139.3

1.92

2.18

RBS-GFP-Term C3

157.6

1.88

2.04

RBS-GFP-Term C4

163.8

1.90

2.19

RBS-GFP-Term C5

155.0

1.91

2.20

DarRoperator PCR: 4 % gel run to check if PCR worked

 

Pouring of 4 % agarose-1xTAE-gel

1. Set waterbath to 70 °C and place gel chamber directly next to it.

2. Take 500 ml bottle (blue lid), add 4 g agarose and fill up to 100 ml with 1xTAE

3. Heat carefully in microwave (check every 5 – 10 sec if agarose does not … äh… überkochen… and mix by shaking bottle slightly (schwenken…) à Dissolving takes just a 2 – 4 min!

4. As soon as agarose is dissolved (clear solution, but many small bubbles from boiling and shaking making it turbid), place it in the waterbath (lid closed), wait for some time until bubbles are gone

5. Check again if agarose is still dissolved, if not, go back to step 3…

6. Pour gel rapidly.

 

Loading of

-          4 µl PCR reaction + 1 µl 5xLD

-          4 µl primer iGEM_36 + 1 µl 5xLD

-          4 µl primer iGEM_37 + 1 µl 5xLD

-          3 µl 2 log ladder (marker)

 

Run at 80 V

EtBr staining; destaining in water

UV detection

 

4% Gel with DarR operator:

Loading:

Lane 1: 2 log ladder; Lane 2: iGEM_36; Lane 3: iGEM_37; Lane 4: Negative control of PCR; Lane 4: PCR reaction tube 1; Lane 5: PCR reaction tube 2; Lane 6: PCR reaction tube 3; Lane 7: 2 log ladder

 

 

è PCR worked

è All reactions were pooled into one reaction

è Determination of concentration and purity (does actually not make sense…)

 

Sample

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

DarRop PCR product

1063.5

1.65

1.11

è Far too much since PCR reaction, that was not purified: Calculate with 100 ng!

 

Restriction Digestion of DarRop PCR product for ligation with pSB1C3 obtained from R.D+gel ex of part 7

-          More or less according to BioBrick Assembly Kit

 

Component

Reaction 1 µl PCR product

Reaction 5 µl PCR product

Reaction 10 µl PCR product

10x NEB2.1 buffer

5 µl

5 µl

5 µl

Eco-RI HF

1 µl

1 µl

1 µl

PstI

1 µl

1 µl

1 µl

PCR product

1 µl

5 µl

10 µl

dH2O

42 µl

38 µl

33 µl

Total

50 µl

50 µl

50 µl

è Reactions with 5 or 10 µl were incubated for 1 h at 37 °C

è Reaction with 1 µl was incubated for >1h at 37 °C

è Heat kill for all reactions: 20 min, 80 °C

 

-          Gel run

-          4 % agarose-1xTAE gel

-          Loading of

     4 µl pf PCR product + 1 µl 5x LD as a control

     8 µl of RD reactions + 2 µl 5x LD à just 8 µl of final sample (10 µl) loaded

     3 µl 2 log ladder as a marker

-          Run at 80 V

-          EtBr staining, destaining in water

-          UV detection

 

Gel:

Loading:

Lane 1: 2 log ladder; Lane 2: DarRop PCR product uncut; Lane 3: DarRop PCR product R.D. 1 µl; Lane 4: DarRop PCR product R.D. 5 µl; Lane 5: DarRop PCR product R.D. 10 µl

è PCR product still detectable for 5 and 10 µl reaction, but not for 1 µl reaction

è For 1 µl reaction probably band at bottom of lane à degradation?

è Use 5 and 10 µl reactions for cloning

è Reactions stored at -20 °C in pink box

 

Pooling of samples from gel ex on 4.7.13

- pSB1C3 vector from part 7

- GFP-Terminator insert

à determination of new concentration with NanoDrop

 

Test gel for plasmids and inserts

- plasmids for part 7 and GFP-Terminator C1 isolated in today’s MiniPrep

- plasmids part 1, 2, 3, 4 (purified on 2.7.13)

- GFP-Terminator insert after gel ex

- pSB1C3 vector obtained from RD and gel ex of part 7

- plasmid GFP-Terminator clone 1 (purified on 2.7.13)

-          1%-agarose-1xTAE gel

-          Loading of around 30 ng (or more) of plasmid DNA (0.5 µl for all samples; exceptions: pSB1C3 vector from part 7 à 2 µl, GFP-Terminator insert à 4 µl, filled up with dH2O to 4 µl (exception: GFP-Terminator insert), then addition of 1 µl 5xLD

-          Marker: 3 µl 2 log ladder

-          Run at 100 V

-          EtBr staining, destaining in water

-          UV detection

 

Gel:

Loading:

Lane 1: 2 log ladder; Lane 2: part 1 uncut, Lane 3: part 2 uncut; Lane 4: part 3 uncut; Lane 5: part 4 uncut; Lane 6: part 7 (from today’s prep); Lane 7: gel ex pSB1C3 from part 7; Lane 8: GFP-Terminator C1 plasmid (former prep); Lane 9: GFP-Terminator C1 plasmid (today’s prep); Lane 10: gel ex GFP-Terminator construct from RD of GFP-Terminator C1 plasmid; Lane 11: 2 log ladder

 

è Part 7 is apparently impure à residual undigested plasmid though gel extraction was done (band of uncut plasmid has same height as pSB1C3 vector…)

 

Test Restriction Digestion of RBS-GFP-Terminator clones 1 – 5

-          Fermentas/ThermoScientific enzymes were used

For 1 reaction:

 

Component

Amount

Plasmid DNA

200 – 300 ng (1 – 1.5 µl in this case)

PstI

0.5 µl

EcoRI

0.5 µl

dH2O

Depends on plasmid amount (7 or 6.5 µl)

10x buffer O

1 µl

Total

10 µl

 

è Preparation of plasmid amount in 3 µl total volume:

Plasmid from…

…C1

…C2

…C3

…C4

…C5

Plasmid

1 µl

1.5 µl

1.5 µl

1.5 µl

1.5 µl

dH2O

2 µl

1.5 µl

1.5 µl

1.5 µl

1.5 µl

è Preparation of MasterMix

Component

Amount

PstI

3 µl

EcoRI

3 µl

dH2O

30 µl

10x buffer O

6 µl

Total

42 µl

 

è Add 7 µl to each reaction

-          Incubation at 37 °C for 1.5 h (no heat kill necessary in this case, since it’s just run on the gel and then thrown away)

-          Addition of 2.5 µl 5xLD

-          Agarose gel electrophoresis

1%-agarose-1xTAE gel

Loading of 10 µl of C1 RD (well run over…), thus loading of 8 µl of other RD’s

Loading of mixture consisting of 0.5 µl of all uncut plasmids + 3.5 µl dH2O + 1 µl 5xLD as controls

Loading of 3 µl 2 log ladder as a marker

-          Run at 100 V

-          EtBr staining, destaining in water

-          UV detection

 

Gel for Test restriction digestion of RBS-GFP-Terminator clones 1 - 5

Loading:

Lane 1: 2 log ladder; Lane 2: C1 uncut; Lane 3: C1 R.D.; Lane 4: C2 uncut; Lane 5: C2 R.D.; Lane 6: C3 uncut; Lane 7: C3 R.D.; Lane 8: C4 uncut; Lane 9: C4 R.D.; Lane 10: C5 uncut; Lane 11: C5 R.D.; Lane 12: 2 log ladder

è Something strange of 100 bp got cloned…

Fold ↑

08th

Mini Plasmid Prep, PCR for DarRoperator, Ligation of Riboswitch inserts

Back-Up-Plates

è For all inoculated RBS-GFP-Terminator clones

è Incubation over day at 37 °C

MiniPlasmidPrep

è Clones 1 – 5 of RBS-GFP-Terminator construct

è Used buffer A4 without Ethanol added… concentrations after elution were quite low…

è Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

Sample

Concentration (ng/µl)

A260nm/A280nm

A260nm/A230nm

Clone 1

4.3

1.92

0.49

Clone 2

3.1

1.53

0.31

Clone 3

1.7

2.74

0.48

Clone 4

3.8

2.09

0.34

Clone 5

4.1

2.31

0.44

è Repeat MiniPrep

PCR for DarRoperator

è As on 19.6.13

Component

1x Reaction

5x Master Mix

5x buffer HF

10 µl

50 µl

PhuS

1 µl

-

Primer iGEM_36

4 µl

20 µl

Primer iGEM_37

4 µl

20 µl

dNTPs

2 µl

145 µl

dH2O

29 µl

10 µl

Total

50 µl

245 µl à add 49 µl to each reaction

Control: 1µl dH2O instead of PhuS polymerase

Protocol:

 

Temperature

Time

No. of cycles

Initial Denaturation

98.5 °C

5min

1

Denaturation

98.5 °C

30s

10 cycles

Annealing

61.0°C

30s

Elongation

72.0°C

2min

Final Elongation

72.0°C

10min

1

Hold

15 °C

1

è Reactions stored at -20°C in 50 ml Falcon tube

Ligation of Riboswitch inserts A, B, C, D with pSB1C3 backbone obtained from gel ex of dig. Part 7 plasmid

 

Ligation more or less according to BioBrick-Assembly-Kit

Ca. 20 ng of pSB1C3 vector ligated with ca. 20 ng of the different inserts

 

Reactions:

Component

RiboA

(iGEM_42/43)

RiboB

(iGEM_41/42)

RiboC

(iGEM_40/43)

RiboD (iGEM_40/41)

DarR

w/o insert control

MasterMix

pSB1C3 vector (tube 3; 10.2 ng/µl)

2 µl

2 µl

2 µl

2 µl

2 µl

2 µl

14 µl

insert

3.5 µl

(5.9 ng/µl)

1.5 µl

(15.1 ng/µl)

2 µl

(11.7 ng/µl)

3 µl

(7.1 ng/µl)

3 µl

(6.8 ng/µl)

-

-

dH2O/HPLC-H2O

11.5 µl

13.5

13 µl

12 µl

12 µl

15 µl

70 µl

Ligase

1 µl

1 µl

1 µl

1 µl

1 µl

1 µl

7 µl

10x ligation buffer

2 µl

2 µl

2 µl

2 µl

2 µl

2 µl

14 µl

Total

20 µl

20 µl

20 µl

20 µl

20 µl

20 µl

105 µl

è Prepare 5 µl of insert-HPLC water mix and add 15 µl of Master Mix

è Incubation for 20 min at RT

è Heat kill for 20 min at 80 °C

Transformation of E.coli DH5α competent cells

-          According to methods folder

             Transformation of

              part7-vector+Ribo A

              part7-vector+Ribo B

              part7-vector+Ribo C

              part7-vector+Ribo D

              part7-vector+DarR

              part7-vector w/o insert

              Control

-          Whole ligation mix used

-          Negative control: 20 µl dH2O

-          Selection on LBCm plates (35 µg/ml Cm)

Inoculation of

RBS-GFP-Term C1, C2, C3, C4, C5

part7 C1

GFP-Terminator C1

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