Team:Stockholm/6 September 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/