Team:Stockholm/6 September 2010

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==Andreas==
==Andreas==
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
====Transformation results====
 +
''From 3/9''
 +
 +
More growth on the 2 μl plate than on the 5 μl plate, indicating what I already suspected: contamination in transformation. Discarded both plates and restarted cloning.
 +
 +
====Digestion====
 +
 +
[N-CPP plasmid] = 672 ng/μl
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!width="50"|N-CPP
 +
|-
 +
|10X buffer Tango
 +
|align="center"|2
 +
|-
 +
|DNA (1 μg)
 +
|align="center"|1.5
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|12.5
 +
|-
 +
|XbaI
 +
|align="center"|1
 +
|-
 +
|AgeI
 +
|align="center"|4
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
|}
 +
 +
:Incubation: 37 &deg;C, 3 h<br />
 +
:Inactivation: 80 &deg;C, 20 min
 +
 +
====Control digestions====
 +
Did two control digestions to analyze the N-CPP plasmid vector size.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="50"|A+S
 +
!width="50"|ApaI
 +
|-
 +
|10X buffer Tango
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|DNA
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|15
 +
|align="center"|16
 +
|-
 +
|FD ApaI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|FD SmaI
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
 +
:Incubation: 37 &deg;C, 30 min<br />
 +
:Inactivation: 65 &deg;C, 5 min
 +
 +
=====Gel verification=====
 +
[[image:Gelver NCPP dig 6sep.png|200px|right|thumb|'''Gel verification of N-CPP plasmid digestions.'''<br />1 kb &lambda; = O'GeneRuler 1 kb DNA ladder; 50 bp &lambda; = GeneRuler 50 bp DNA ladder.]]
 +
1 % agarose, 120 V, 40 min
 +
 +
*'''N-CPP:''' undigested N-CPP plasmid
 +
*'''Dig X+A:''' N-CPP plasmid digested with XbaI & AgeI
 +
*'''Dig ApaI:''' Linearized N-CPP plasmid digested with ApaI
 +
*'''Dig S+A:''' N-CPP plasmid digested with SmaI and ApaI, excising the 391 bp N-CPP cluster.
 +
 +
'''Results'''<br />
 +
Linearized plasmid seems to be &asymp;5 kb long. Undigested plasmid moved slower; this was probably the result of the lane being too crowded.
 +
 +
====Ligation====
 +
 +
Vector: Dig pSB1C3 X+A EXTR 1
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="50"|1
 +
!width="50"|2*
 +
|-
 +
|5X Rapid Ligation buffer
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|Vector DNA
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|Insert DNA
 +
|align="center"|11
 +
|align="center"|9
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|align="center"|2
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!22 &mu;l &dagger;
 +
!20 &mu;l
 +
|}
 +
&dagger; ''Accidentally added 2 &mu;l FastAP alkaline phosphatase to ligation mix 1. Enzyme inactivated at 75 &deg;C, 10 min, prior to ligation.''
 +
*Incubation: 22 &deg;C, 10 min
 +
 +
====Transformation====
 +
''Performed by Mimmi''
 +
 +
Standard transformation, except:
 +
*30 min incubation in 37 &deg;C
 +
*3 &mu;l ligation mix
 +
Cells grown on Cm 25 plates.
 +
 +
====PCR====
 +
Since we've experienced some problems with our N-CPP clonings, a PCR of each individual N-CPP was performed for individual cloning, in case the current cloning doesn't work.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|Reactions & primers
 +
|-
 +
|&nbsp;
 +
!Forward
 +
!Reverse
 +
|-
 +
|'''N-Tra10'''
 +
|align="center"|VF2
 +
|align="center"|VR
 +
|-
 +
|'''N-TAT'''
 +
|align="center"|pEXf
 +
|align="center"|pEXr
 +
|-
 +
|'''N-LMWP'''
 +
|align="center"|pGexf
 +
|align="center"|pGexr
 +
|-
 +
|'''N-CPP cluster'''
 +
|align="center"|VF2
 +
|align="center"|pGexr
 +
|}
 +
<br />
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="2"|PCR tubes
 +
|-
 +
|10X Pfu buffer
 +
|align="center" width="50"|5
 +
|-
 +
|dNTP, 10 mM
 +
|align="center"|1
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|41
 +
|-
 +
|Template DNA
 +
|align="center"|0.5
 +
|-
 +
|Forw. primer
 +
|align="center"|1
 +
|-
 +
|Rev. primer
 +
|align="center"|1
 +
|-
 +
|Pfu DNA pol.
 +
|align="center"|0.5
 +
|-
 +
|&nbsp;
 +
!50 &mu;l
 +
|}
 +
 +
Standard colony PCR settings
 +
*Elongation: 1:15
 +
 +
Tubes stored in -20 &deg;C.
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:00, 26 October 2010

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Contents

Mimmi

MITF-M

Gel

2010-09-06 MITF-M.jpg
well sample
1 1kb ladder
2 MITF-M 1
3 MITF-M 2
4 MITF-M 3
5 MITF-M 4
6 positive control
7 blank

Andreas

Cloning of N-CPPs into pSB1C3

Transformation results

From 3/9

More growth on the 2 μl plate than on the 5 μl plate, indicating what I already suspected: contamination in transformation. Discarded both plates and restarted cloning.

Digestion

[N-CPP plasmid] = 672 ng/μl

  N-CPP
10X buffer Tango 2
DNA (1 μg) 1.5
dH2O 12.5
XbaI 1
AgeI 4
  20 μl
Incubation: 37 °C, 3 h
Inactivation: 80 °C, 20 min

Control digestions

Did two control digestions to analyze the N-CPP plasmid vector size.

  A+S ApaI
10X buffer Tango 2 2
DNA 1 1
dH2O 15 16
FD ApaI 1 1
FD SmaI 1 0
  20 μl 20 μl
Incubation: 37 °C, 30 min
Inactivation: 65 °C, 5 min
Gel verification
Gel verification of N-CPP plasmid digestions.
1 kb λ = O'GeneRuler 1 kb DNA ladder; 50 bp λ = GeneRuler 50 bp DNA ladder.

1 % agarose, 120 V, 40 min

  • N-CPP: undigested N-CPP plasmid
  • Dig X+A: N-CPP plasmid digested with XbaI & AgeI
  • Dig ApaI: Linearized N-CPP plasmid digested with ApaI
  • Dig S+A: N-CPP plasmid digested with SmaI and ApaI, excising the 391 bp N-CPP cluster.

Results
Linearized plasmid seems to be ≈5 kb long. Undigested plasmid moved slower; this was probably the result of the lane being too crowded.

Ligation

Vector: Dig pSB1C3 X+A EXTR 1

  1 2*
5X Rapid Ligation buffer 4 4
Vector DNA 4 4
Insert DNA 11 9
dH2O 0 2
T4 DNA ligase 1 1
  22 μl † 20 μl

† Accidentally added 2 μl FastAP alkaline phosphatase to ligation mix 1. Enzyme inactivated at 75 °C, 10 min, prior to ligation.

  • Incubation: 22 °C, 10 min

Transformation

Performed by Mimmi

Standard transformation, except:

  • 30 min incubation in 37 °C
  • 3 μl ligation mix

Cells grown on Cm 25 plates.

PCR

Since we've experienced some problems with our N-CPP clonings, a PCR of each individual N-CPP was performed for individual cloning, in case the current cloning doesn't work.

Reactions & primers
  Forward Reverse
N-Tra10 VF2 VR
N-TAT pEXf pEXr
N-LMWP pGexf pGexr
N-CPP cluster VF2 pGexr


PCR tubes
10X Pfu buffer 5
dNTP, 10 mM 1
dH2O 41
Template DNA 0.5
Forw. primer 1
Rev. primer 1
Pfu DNA pol. 0.5
  50 μl

Standard colony PCR settings

  • Elongation: 1:15

Tubes stored in -20 °C.





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

Retrieved from "http://2010.igem.org/Team:Stockholm/6_September_2010"