Team:Goettingen/NoteBook w3

From 2013.igem.org

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<p class="timeline-title goe-rt" title="Click to fold/extend">Digestion of PCR product (DarR Operator) (10 cycles) and Plasmid backbone (pSB1A3)</p>
<p class="timeline-title goe-rt" title="Click to fold/extend">Digestion of PCR product (DarR Operator) (10 cycles) and Plasmid backbone (pSB1A3)</p>
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   <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Concentration
   <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Concentration
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<span class="date">19th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
<span class="date">19th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
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<p class="timeline-title goe-rt" title="Click to fold/extend">PCR using primer iGEM 36,37 for construction of DarR operator biobrick,DarR sequencing</p>
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<p class="timeline-title goe-rt">PCR using primer iGEM 36,37 for construction of DarR operator biobrick,DarR sequencing</p>
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   <p class="MsoNormal"><b><span lang="EN-US">PCR using primer iGEM 36<span class="GramE">,37</span> for construction of DarR operator biobrick.<o:p></o:p></span></b></p>
   <p class="MsoNormal"><b><span lang="EN-US">PCR using primer iGEM 36<span class="GramE">,37</span> for construction of DarR operator biobrick.<o:p></o:p></span></b></p>
   <p class="MsoNormal"><span lang="EN-US">Same recipe as yesterdays PCR, rest the
   <p class="MsoNormal"><span lang="EN-US">Same recipe as yesterdays PCR, rest the

Latest revision as of 19:55, 30 September 2013

21st

Transformation from yesterday worked!

Transformation from yesterday worked! 6 clones! And the Without-Insert-Control lacks clones!

è     Plates stored at 4 °C


20th

Digestion of PCR product (DarR Operator) (10 cycles) and Plasmid backbone (pSB1A3)

Concentration of stored PCR products from 19.6.13

No. of PCR cycles

c(DNA) [ng/µl]

A260/A280

A260/A230

PCR product

10 cycles

1068.7

1.63

1.07

PCR product

20 cycles

975.7

1.66

1.10

PCR product

30 cycles

964.9

1.65

1.11

 

Digestion of PCR product (AND WHATS IN THE PCR?) (10 cycles) and Plasmid backbone (pSB1A3 (with AmpR))

According to: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones

 

Ezyme master mix:

5 µl Buffer 0

0.5 µl EcoRI

0.5 µl PstI

18 µl dH2O

 

Digestion of backbone resp. insert:

4 µl linearized plasmid backbone (25 ng/µl for 100 ng total)

4 µl Enzyme master mix

resp.:

5 µl insert

4 µl Enzyme master mix

resp.:

10 µl insert

4 µl Enzyme master mix

 

digest 37°C/30 min heat kill 80°C/20 min

 

Ligation of backbone and insert:

According to: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones

 

2 µl digested plasmid backbone

10 µl EcoRI and PstI digested fragment

1.5 µl T4 DNA Ligase buffer

0.5 µl T4 DNA Ligase

1 µl H2O

ligate 30°C/30 min heat kill 80°C/min

 

Transformation of ligated parts

According to method book

Digestion of amplified DAC-domains (S.aureus and S. pneumonia) from 17.06.

Digestion of amplified DAC-domains (S.aureus and S. pneumonia) from 17.06.

 

Components:

Volume

 

plasmid DNA

 

16µl

PstI

6µl

EcoRI

6µl

orange buffer (10x)

8µl

H2O

44µl

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

 

Digestion of prepared Strep-Tag from 17.06.

 

Components:

Volume

Strep-Tag

16µl

PstI

6µl

EcoRI

6µl

orange buffer (10x)

8µl

H2O

44µl

 

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

 

è Preparation of Strap-Tag worked

è Preparation of DAC domain of S.aureus worked

è Preparation of DAC domain of S- pneumonia failed = EcoRI restriction site in fragment

 

Fold ↑

19th

PCR using primer iGEM 36,37 for construction of DarR operator biobrick,DarR sequencing

PCR using primer iGEM 36,37 for construction of DarR operator biobrick.

Same recipe as yesterdays PCR, rest the protocol,

98.5

5min

 

 

 

98.5

30s

10 cycle

20cycle

30cycle

61.0

30s

72.0

2min

72.0

10min

 

 

 

 Ctrl: no enzyme

Increase the gel concentration to 4%.


PCR worked, Gel here:

Lane 1-8: ladder, iGEM36, iGEM37, ctrl(no Polyermase), 1 cycle, 10cycle, 20cycle, 30cycle.

Gel concentration : 4%

1cycle PCR product from yesterday.

So, use 4% gel, 10cycle PCR is enough


DarR sequencing results

in “our” genomic DNA of M. smegmatis, there are two additional codons inserted right after the GTG start codon:

“our” gDNA: GTG  AGC  GCG  AGC  GCC à Ser-Ala-Ser-Ala

Paper gDNA: GTG  _ _ _  _ _ _  AGC  GCC à Ser-Ala

Additionally, there are 5 point mutations:

120 C à G

132 A à G

210 T à C

228 C à T

263 T à C

All are silent, except for one: aa 87 Val à Ala (both small, hydrophobic aa)


Primer Design

Design of new DarR biobrick fwd primer (since 2 additional codons inserted and old primer designed for DarR gene without these codons)


18th

Test restriction of Part 2 C2,Primer design,Construction of DarR operator biobrick(PCR)

Plate reader assay

-          Plate thrown away after ON measurement

-          Results:

 

Concentration of plasmid purified from Clone 2 of Part 2

Part no.

c(DNA)

[ng/µl]

A260/A280

A260/A230

2 C2

120.6

1.87

2.24

 

Test restriction of Part 2 Clone 2

Component

Uncut control

SpeI single digestion

PstI single digestion

SpeI/PstI double digestion

Plasmid DNA (150 ng)

1.2 µl

1.2 µl

1.2 µl

1.2 µl

10x buffer

1 µl (Tango)

1 µl (Tango)

1 µl (buffer 0)

1 µl (Tango)

SpeI

-

1 µl

-

1 µl

PstI

-

-

1 µl

2 µl

dH2O

7.8 µl

6.8 µl

6.8 µl

4.8 µl

Total

10 µl

10 µl

10 µl

10 µl

-          Incubation for ca. 1.5 h at 37 °C (incubator)

-          Addition of 2 µl of 5x DNA loading dye and run on 1 % 1xTAE-agarose gel (marker: 100 bp ladder)

-          EtBr staining and destaining in H2O, UV detection

è     Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

Conclusions:

è     Clone 2 contains the expected plasmid harboring part 2 (impure stock?)

 

Primer design and ordering

Design of primer iGEM_43

Ordering of primers iGEM_38 to iGEM_43 from Sigma-Aldrich

 

PCR:

PCR using primer iGEM32,33 for construction of DarR biobrick.

Because of those two primers have high tendency to form 2nd structure, 1x 2x 4x primers are applied, to see the recipe, see dropbox > Reporter Team > PCR_iGEM32_33.xls

PCR was successful, better use 2x primer. Protocol as follows:

98.5

5min

 

98.5

30s

30 cycles

67.0

35s

72.0

2min

72.0

10min

 

 

è     Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

PCR using primer iGEM 36,37 for construction of DarR operator biobrick. To see the recipe, see dropbox > Reporter Team > PCR_iGEM32_33.xls

Protocol as follows:

98.5

5min

Only one cycle

98.5

30s

61.0

30s

72.0

12min

 

è     Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

 

2% gel cannot separate the primers and PCR product:

iGEM36:36nt

iGEM37:35nt

PCR product should be 57bp

Gel1: forget the primer as ctrl

Gel2: load the wrong primer

 


17th

Plate reader assay for the pink strains, Plasmid mini prep for Part C2

Cryo-Stocks of E. coli transfomant (2, C2)

Stored in -70 °C (red box)

 

Plasmid Mini-Preparation of part 2 C2

-          harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation à stored at – 20 °C in red box

-          harvesting of remaining culture for today’s prep

è     Performed as on 7.6.13

 

Plate reader assay

-          Measurment of OD of ON cultures in 1:10 dilution in LB; LB as blank (see table)

-          Preparation of E.coli cultures of an OD600nm = 0.05 in LB (without antibiotics) in 96-well microtiterplate;

final volume: 180 µl (see table) à triplicates for each clone

Calculation:

V(well culture) * OD600nm (wanted)/ OD600nm (ON-culture) = V(ON culture needed for preparation of well cultures)

180 µl * 0.05/OD600nm (ON-culture) = V(ON culture needed for preparation of well cultures) [µ]

 

Values and amounts for well cultures:

Clone

OD600nm

(ON culture)

1:10

OD600nm

(ON culture)

1:1

V(ON culture for preparation of well culture)[µ]

DHα

0.433

4.33

2.1

Part 1

C1

0.426

4.26

2.1

C2

0.450

4.50

2

Part 2

C1

0.416

4.16

2.2

C2

0.436

4.36

2.1

C3

0.434

4.34

2.1

Part 3

C1

0.467

4.67

1.9

C2

0.475

4.75

1.9

C3

0.473

4.73

1.9

Part 4

C1

0.457

4.57

2

C 2

0.466

4.66

1.9

C3

0.437

4.37

2.1

Part 6

C1

0.450

4.50

2

C2

0.431

4.31

2.1

C3

0.448

4.48

2

è     Pipetting of ON-culture volume into each well, then addition of 180 µl LB without antibiotics using multi-channel pipett (be careful: tips must be fixed properly!)

è     For scheme: see folder

-          Incubation of plate at 37 °C with vortexing in plate reader, fluorescence of GFP and RFP, as well as OD600nm measured every 15 min for approx. 3 h

 

Fluorescence excited and measured at:

 

Absorption

Emission

RFP

555 nm

583 nm

GFP

489 nm

509 nm

-          Storage of plate at 4 °C

-          Measurement ON under same conditions

Preperation of Strep-Tag for Biobrick assembly

Preperation of Strep-Tag for Biobrick assembly

 

Components:

Volume (µl)

Primer iGEM_11 (5µM)

5µl

Primer iGEM_12 (5µM)

5µl

dNTPs

1µl

buffer (5x)

10µl

H2O

28µl

 

è incubation at 90°C for 5min, cool down at room temperature and add 1µl of PfuS polymerase

è incubation at 72°C for 15min

 

Amplification of S. aureus and S. pneumonia DAC domains for Biobrick assembly

 

Components:

Volume

Primer iGEM_13 (5µM)

2µl

Primer iGEM_14 (5µM)

2µl

Primer iGEM_15 (5µM)

2µl

Primer iGEM_16 (5µM)

2µl

genomic DNA

2µl

dNTPs

2µl

buffer (5x)

10µl

H2O

33µl

PfuS polymerase

1µl

 

Fold ↑

16th

Innoculation of Part 3 C2