Team:Stockholm/7 September 2010

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==Andreas==
==Andreas==
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
 +
====Transformation results====
 +
''From 6/9 transformations''
 +
 +
Good colony yields on both plates, 1 and 2*.
 +
 +
====Colony PCR====
 +
4 colonies picked from each plate for verification by colony PCR.
 +
*1, 2, 3, 4, 5*, 6*, 7*, 8*
 +
*PC: pSB1C3.RFP
 +
 +
{|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
 +
|-
 +
|dNTP, 10 mM
 +
|align="center"|0.4
 +
|-
 +
|Fwd primer (VF2)
 +
|align="center"|0.4
 +
|-
 +
|Rev primer (VR)
 +
|align="center"|0.4
 +
|-
 +
|Cell suspension
 +
|align="center"|0.5
 +
|-
 +
|DreamTaq pol.
 +
|align="center"|0.08
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
|}
 +
 +
Standard colony PCR settings
 +
*Elongation time: 0:45
 +
 +
====Gel verification====
 +
[[image:ColPCR_pC.N-CPP_7sep.png|200px|thumb|right|'''Colony PCR gel verification of pSB1C3.N-CPPs'''<br />3 &mu;l &lambda;; 6 &mu;l sample.<br />&lambda; = GeneRuler 50 bp DNA ladder.]]
 +
1.5 % agarose, 90 V.
 +
 +
'''Expected bands'''
 +
*pSB1C3.Tra10: 389 bp
 +
*pSB1C3.TAT: 359 bp
 +
*pSB1C3.LMWP: 368 bp
 +
 +
'''Results'''<br />
 +
Relevant bands in clones 3, 4, 6, 7 & 8. Slightly different in size, which could indicate presence of all three N-CPPs. All clones selected for sequencing.
 +
 +
====ON cultures====
 +
''For plasmid prep''
 +
*pSB1C3.N-CPP: pC.NCPP 3, 4, 6*, 7* and 8*
 +
**5 ml LB + Cm 25
 +
**37 &deg;C, 200 rpm ON
 +
 +
===Transfer of m-yCCS into pEX===
 +
====ON cultures====
 +
Set ON cultures of pEX.yCCS for plasmid prep and glycerol stocks. From 3/9 colonies.
 +
*pEX.yCCS 5 and 8
 +
**5 ml LB + Amp 100
 +
**37 &deg;C, 200 rpm ON
 +
*pEX.yCCS 5 and 8
 +
**5 ml LB + Amp 100
 +
**30 &deg;C

Revision as of 14:43, 8 September 2010

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Contents

Andreas

Cloning of N-CPPs into pSB1C3

Transformation results

From 6/9 transformations

Good colony yields on both plates, 1 and 2*.

Colony PCR

4 colonies picked from each plate for verification by colony PCR.

  • 1, 2, 3, 4, 5*, 6*, 7*, 8*
  • PC: pSB1C3.RFP
PCR tubes
dH2O 16.22
DreamTaq buffer 2
dNTP, 10 mM 0.4
Fwd primer (VF2) 0.4
Rev primer (VR) 0.4
Cell suspension 0.5
DreamTaq pol. 0.08
  20 μl

Standard colony PCR settings

  • Elongation time: 0:45

Gel verification

Colony PCR gel verification of pSB1C3.N-CPPs
3 μl λ; 6 μl sample.
λ = GeneRuler 50 bp DNA ladder.

1.5 % agarose, 90 V.

Expected bands

  • pSB1C3.Tra10: 389 bp
  • pSB1C3.TAT: 359 bp
  • pSB1C3.LMWP: 368 bp

Results
Relevant bands in clones 3, 4, 6, 7 & 8. Slightly different in size, which could indicate presence of all three N-CPPs. All clones selected for sequencing.

ON cultures

For plasmid prep

  • pSB1C3.N-CPP: pC.NCPP 3, 4, 6*, 7* and 8*
    • 5 ml LB + Cm 25
    • 37 °C, 200 rpm ON

Transfer of m-yCCS into pEX

ON cultures

Set ON cultures of pEX.yCCS for plasmid prep and glycerol stocks. From 3/9 colonies.

  • pEX.yCCS 5 and 8
    • 5 ml LB + Amp 100
    • 37 °C, 200 rpm ON
  • pEX.yCCS 5 and 8
    • 5 ml LB + Amp 100
    • 30 °C
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