Team:Stockholm/1 September 2010

From 2010.igem.org


Contents

Andreas

His⋅SOD cloning into pMA

Digestion

[pSB1C3.m-SOD]=105.5 ng/μl
[pMA.His]=85.67 ng/μl

[μl] SOD pMA.His
10X FD buffer 3 3
DNA (2 μg) 19 23
dH2O 6 3
FD AgeI 0 0.5
NgoMIV 1 0
FD SpeI 1 1
  30 30.5

Incubation:

  • 37 °C, 0:30 (FD)
  • 37 °C, 2:00 (NgoMIV)

Inactivation:

  • 80 °C, 10 min

Ligation

  [μl]
5X Rapid ligation buf. 4
Vector DNA (pMa.His) 1.5
Insert DNA (m-SOD) 4.5
dH2O 9
T4 DNA ligase 1
  20

Incubation:

  • 22 °C, 10 min

Transformation

Quick-transformation according to protocol into both Top10 and DH5α to compare and verify Top10 competence:

  • 1.5 μl
  • Amp 100

SOD⋅His cloning into pMA

Glycerol stocks

From 31/8 ON cultures

  • SH1: pMA.SOD⋅His 1, 1/9/2010
  • SH2: pMA.SOD⋅His 2, 1/9/2010

Transfer of m-yCCS into pEX

Digestion

[pEX.RFP] = 82 ng/μl [pSB1C3.m-yCCS] = 101.2 ng/μl

[μl] SOD pMA.His
10X FD buffer 3 3
DNA (1 μg) 12 6
dH2O 13 19
FD XbaI 1 1
FD PstI 1 1
  30 30

Incubation:

  • 37 °C, 10 min

Inactivation:

  • 65 °C, 20 min

Ligation

  [μl]
5X Rapid ligation buf. 4
Vector DNA (pEX) 1.5
Insert DNA (m-yCCS) 9
dH2O 4.5
T4 DNA ligase 1
  20

Incubation:

  • 22 °C, 10 min

Transformation

Quick-transformation (Top10)

  • 2 μl
  • Amp 100


Mimmi

MITF-M, starting over...

Colony PCR

Mix (µl) Primers Conditions
sH2O 22.5 MITF_FB_F_18aug time °C
F primer 1 MITF_FB_R_18aug 2m 95
R Primer 1 30s 95 )
DNA 0.5 30s 55 > 5 cycles
tot 25µl 1m40s 72 )
30s 95 \ 25 cycles
2m10s 72 /
10m 72
oo 10


Gel

2010-09-01 MITF-M + FB.jpg
well sample
1 1kb ladder
2 MITF-M + FB









Digestion

Mix (µl) (µl) Conditions
MITF/pSB1C3 22 20 Time °C
s2O 10 5 30m 37
10x buffer 4 3 20m 65
EcoRI 2 1 oo 10
SpeI 2 1
tot 40µl 30µl


Ligation

Mix (µl) Conditions
pSB1C3 3 Time °C
MITF-M 10 10m 22
5x buffer 4 oo 4
T4 ligase 1
s2O 2
tot 20µl





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