Team:Stockholm/10 September 2010

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**3 ml LB + 100 Amp
**3 ml LB + 100 Amp
***30 °C
***30 °C
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===Extraction of RBS BioBrick (BBa_B0034)===
 +
After studying the original RBS of our expression vector pEX, we decided that BBa_B0034 was a better candidate for our SOD/yCCS operon than BBa_B0030, as it better resembles the RBS of pEX, as well as minimizes the distance from the first gene in the operon.
 +
 +
Extracted BBa_B0034 (RBS 34), carried on pSB1A2, from iGEM plate 1, well 2M. Transformed into Top10.
 +
*Quick transformation
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*1 μl DNA
 +
*Amp 100
===Preparation of chemically competent Top10===
===Preparation of chemically competent Top10===
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Plate grown ON in 37 °C.
Plate grown ON in 37 °C.
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== Mimmi ==
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=== Restriction enzymes control ===
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==== Digestion ====
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{|
 +
! mix
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| (µl)
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| rowspan="6" width="100" |
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! colspan="2" | Conditions
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|-
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| sH<sub>2</sub>O
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| 15
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! time
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! &deg;C
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|-
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| 10x buffer
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| 3
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| 30m
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| 37
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|-
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| DNA (pSB1C3)
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| 10
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| 20m
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| 65
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|-
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| enzyme (E/S)
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| 1
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| OO
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| 10
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| align="right" | tot
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| 29µl
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| colspan="2" |
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|}
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==== Gel ====
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[[Image:place_for_picture.jpg|200px|thumb|left|]]
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{|
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! well
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! sample
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|-
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| 1
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| ladder
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|-
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| 2
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| pSB1C3 cut E
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|-
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| 3
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| pSB1C3 cut S
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|}
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=== MITF-M ===
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==== Site-Directed Mutagenesis ====
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*Add Dpn1 (12.30-16=3.5h)
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*Deactivate Dpn1 at 80&deg;C for 20m
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 +
{{Stockholm/Footer}}

Latest revision as of 11:02, 26 October 2010

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Contents

Andreas

Cloning of N-CPPs into pSB1C3 & Extraction of RBS BioBrick (BBa_B0030)

Transformations from 9/9 resulted in good colony yields on all plates. Chose "pSB1C3.N-CPP* 9/9" for colony PCR.

Colony PCR

Picked 12 N-CPP* clones (NC 1-12) and 4 RBS clones (RBS 1-4)

PCR tubes
dH2O 16.22
DreamTaq buffer 2
dNTPs, 10 mM 0.4
VF2 0.4
VR 0.4
DreamTaq pol. 0.08
Template DNA 0.5
  20 μl

PCR settings
Standard colony PCR protocol.

  • 1:00 elongation

Gel verification

Colony PCR gel verification of pSB1C3.N-CPP (NC) and pSB1A2.BBa_B0030 (RBS) clones.
50 bp λ = GeneRuler 50 bp DNA ladder; 1 kb λ = O'GeneRuler 1 kb DNA ladder

Also ran two samples for Mimmi (E & S)

1.5 % agarose, 90 V

Expected bands

  • N-Tra10: 389 bp
  • N-TAT: 359 bp
  • N-LMWP: 368 bp
  • RBS B0030: 253 bp

Results

  • N-CPPs: Potentially correct bands for clones 2, 3, 5, 8, 9, 10, 11 & 12
  • RBS B0030: Correct-sized bands for all four clones.

ON cultures

Set ON cultures for all relevant N-CPPs, for plasmid prep.

  • N-CPP 2, 3, 5, 8, 9, 10, 11, 12
    • 5 ml LB + 25 Cm
    • 37 °C, 220 rpm

Selected clone 4 of RBS 30. Both plasmid prep and glycerol stock.

  • RBS 30 4
    • 5 ml LB + 100 Amp
      • 37 °C, 220 rpm.
    • 3 ml LB + 100 Amp
      • 30 °C

Extraction of RBS BioBrick (BBa_B0034)

After studying the original RBS of our expression vector pEX, we decided that BBa_B0034 was a better candidate for our SOD/yCCS operon than BBa_B0030, as it better resembles the RBS of pEX, as well as minimizes the distance from the first gene in the operon.

Extracted BBa_B0034 (RBS 34), carried on pSB1A2, from iGEM plate 1, well 2M. Transformed into Top10.

  • Quick transformation
  • 1 μl DNA
  • Amp 100

Preparation of chemically competent Top10

Since I've previously experienced slight AmpR contamination in our latest batch of competent Top10, I streaked an LB agar plate with competent Top10 cells to isolate single clones.

Plate grown ON in 37 °C.




Mimmi

Restriction enzymes control

Digestion

mix (µl) Conditions
sH2O 15 time °C
10x buffer 3 30m 37
DNA (pSB1C3) 10 20m 65
enzyme (E/S) 1 OO 10 tot 29µl



Gel

Place for picture.jpg
well sample
1 ladder
2 pSB1C3 cut E
3 pSB1C3 cut S









MITF-M

Site-Directed Mutagenesis

  • Add Dpn1 (12.30-16=3.5h)
  • Deactivate Dpn1 at 80°C for 20m





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

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