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
 +
 +
===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.

Revision as of 09:59, 13 September 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

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.

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