Team:Stockholm/10 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==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)
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="2"|PCR tubes
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center" width="50"|16.22
 +
|-
 +
|DreamTaq buffer
 +
|align="center"|2
 +
|-
 +
|dNTPs, 10 mM
 +
|align="center"|0.4
 +
|-
 +
|VF2
 +
|align="center"|0.4
 +
|-
 +
|VR
 +
|align="center"|0.4
 +
|-
 +
|DreamTaq pol.
 +
|align="center"|0.08
 +
|-
 +
|Template DNA
 +
|align="center"|0.5
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
|}
 +
 +
'''PCR settings'''<br />
 +
Standard colony PCR protocol.
 +
*1:00 elongation
 +
 +
====Gel verification====
 +
[[image:ColPCR_NCPP_RBS30_10sep.png|200px|thumb|right|'''Colony PCR gel verification of pSB1C3.N-CPP (NC) and pSB1A2.BBa_B0030 (RBS) clones.'''<br />50 bp &lambda; = GeneRuler 50 bp DNA ladder; 1 kb &lambda; = 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 &deg;C, 220 rpm
 +
 +
Selected clone 4 of RBS 30. Both plasmid prep and glycerol stock.
 +
*RBS 30 4
 +
**5 ml LB + 100 Amp
 +
***37 &deg;C, 220 rpm.
 +
**3 ml LB + 100 Amp
 +
***30 &deg;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 &deg;C.
 +
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== Restriction enzymes control ===
 +
 +
==== Digestion ====
 +
 +
{|
 +
! mix
 +
| (µl)
 +
| rowspan="6" width="100" |
 +
! colspan="2" | Conditions
 +
|-
 +
| sH<sub>2</sub>O
 +
| 15
 +
! time
 +
! &deg;C
 +
|-
 +
| 10x buffer
 +
| 3
 +
| 30m
 +
| 37
 +
|-
 +
| DNA (pSB1C3)
 +
| 10
 +
| 20m
 +
| 65
 +
|-
 +
| enzyme (E/S)
 +
| 1
 +
| OO
 +
| 10
 +
| align="right" | tot
 +
| 29µl
 +
| colspan="2" |
 +
|}
 +
 +
 +
 +
 +
==== Gel ====
 +
 +
[[Image:place_for_picture.jpg|200px|thumb|left|]]
 +
{|
 +
! 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&deg;C for 20m
 +
 +
{{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/

Retrieved from "http://2010.igem.org/Team:Stockholm/10_September_2010"