Team:Stockholm/8 September 2010

From 2010.igem.org


Contents

Andreas

Transfer of yCCS into pEX

Glycerol stocks

From 7/9 ON cultures

  • pEX.yCCS 5 2010-09-08
  • pEX.yCCS 8 2010-09-08

Plasmid prep

From 7/9 ON cultures
Elution: 70 μl x2

Sample Conc [ng/μl] A260/A280
pEX.yCCS 5 56.32 2.02
pEX.yCCS 8 57.63 1.97

Cloning of N-CPPs into pSB1C3

Plasmid prep

From 7/9 ON cultures
Elution: 70 μl x2

Sample Conc [ng/μl] A260/A280
pSB1C3.N-CPP 3 105.6 1.94
pSB1C3.N-CPP 4 88.87 2.03
pSB1C3.N-CPP 6* 86.64 1.98
pSB1C3.N-CPP 7* 75.79 1.90
pSB1C3.N-CPP 8* 52.64 1.99

Sequencing

15 μl plasmid DNA + 1.5 μl VF2 primer.

  • NCPP 3: ASB0045 B41
  • NCPP 4: ASB0045 B42
  • NCPP 6: ASB0045 B43
  • NCPP 7: ASB0045 B44
  • NCPP 8: ASB0045 B45

Construction and cloning of MITF

MITF PCR

PCR tubes (MITF 1 & MITF 2)
dH2O 41
Pfu buffer 5
dNTPs, 10 mM 1
Fwd primer
(MITF_FB_F_18aug)
1
Rev primer
(MITF_FB_R_18aug)
1
Template DNA
(pRC/CMV MITF-M, 10 ng/μl)
0.5
Pfu polymerase 0.5
  50 μl
PCR settings
95 °C 10:00
95 °C 0:30 x5
55 °C 0:30
72 °C 1:40
95 °C 0:30 x25
72 °C 2:10
72 °C 10:00

Gel verification

1 % agarose, 110 V

  • 3 μl DNA ladder (O'GeneRuler 1 kb DNA ladder
  • 5 μl sample

No gel image available

Results
No visible bands for neither of the two MITF samples.

Colony PCRs

Picked colonies from Mimmi's plates:

  • pSB1C3.MITF (2/9)
    • MITF 1-8
  • pSB1C3.SOD⋅His (6/9)
    • pC.SH 1-3
  • pSB1C3.His⋅SOD (6/9)
    • pC.HS 1-3
  • pEX.His⋅SOD (6/9)
    • pEX.SH 1-3
  • pEX.SOD⋅His (6/9)
    • pEX.HS 1-3

Primers

  • pSB1C3: VF2 & VR
  • pEX: pEXf & pEXr
PCR tubes
dH2O 16.22
DreamTaq buffer 2
dNTPs, 10 mM 0.4
DreamTaq pol. 0.08
Fwd primer 0.4
Rev primer 0.4
Cell susp. (template) 0.5
  20 μl
PCR settings
95 °C 3:00
95 °C 0:30 x30
55 °C 0:30
72 °C 1:00
72 °C 5:00

Gel verification

Colony PCR gel verification of pSB1C3.MITF, pSB1C3.SOD*His/.His*SOD and pEX.SOD*His/.His*SOD.
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 90 V, 50 min

Expected bands

  • MITF: 1586 bp
  • pC.SH/pC.HS: 815 bp
  • pEX.SH/pEX.HS: 702 bp

Results
All MITF samples resulted in ≈300-350 bp bands, which corresponds to re-ligated pSB1C3 vector (316 bp). This is strange, since re-ligation should not be possible.
All other samples correspond very well to expected sizes. pC.SH3, pC.HS1, pEX.SH1 & pEX.HS1 were selected for plasmid prep and glycerol stocks.

ON cultures

  • 5 ml LB + 25 Cm; 37 °C, 225 rpm
    • pC.SH3
    • pC.HS3
  • 5 ml LB + 100 Amp; 37 °C, 225 rpm
    • pEX.SH1
    • pEX.HS1
  • 3 ml LB + 25 Cm; 30 °C
    • pC.SH3
    • pC.HS3
  • 3 ml LB + 100 Amp; 30 °C
    • pEX.SH1
    • pEX.HS1

Hassan

Animation





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