Team:Stockholm/1 September 2010

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==Andreas==
==Andreas==
 +
 +
===His⋅SOD cloning into pMA===
 +
 +
====Digestion====
 +
 +
[pSB1C3.m-SOD]=105.5 ng/&mu;l<br />
 +
[pMA.His]=85.67 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|align="center"|[&mu;l]
 +
!width="50"|SOD
 +
!width="50"|pMA.His
 +
|-
 +
|10X FD buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|DNA (2 &mu;g)
 +
|align="center"|19
 +
|align="center"|23
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|6
 +
|align="center"|3
 +
|-
 +
|FD AgeI
 +
|align="center"|0
 +
|align="center"|0.5
 +
|-
 +
|NgoMIV
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|FD SpeI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!30
 +
!30.5
 +
|}
 +
 +
'''Incubation:'''
 +
*37 &deg;C, 0:30 (FD)
 +
*37 &deg;C, 2:00 (NgoMIV)
 +
 +
'''Inactivation:'''
 +
*80 &deg;C, 10 min
 +
 +
====Ligation====
 +
 +
{|border="1" cellpadding="1" cellspacing="1"
 +
|&nbsp;
 +
|align="center"|[&mu;l]
 +
|-
 +
|5X Rapid ligation buf.
 +
|width="40" align="center"|4
 +
|-
 +
|Vector DNA (pMa.His)
 +
|align="center"|1.5
 +
|-
 +
|Insert DNA (m-SOD)
 +
|align="center"|4.5
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|9
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20
 +
|}
 +
 +
'''Incubation:'''
 +
*22 &deg;C, 10 min
 +
 +
====Transformation====
 +
Quick-transformation according to protocol into both Top10 and DH5&alpha; to compare and verify Top10 competence:
 +
*1.5 &mu;l
 +
*Amp 100
 +
 +
===SOD&sdot;His cloning into pMA===
 +
 +
====Glycerol stocks====
 +
''From 31/8 ON cultures''
 +
 +
*SH1: pMA.SOD&sdot;His 1, 1/9/2010
 +
*SH2: pMA.SOD&sdot;His 2, 1/9/2010
 +
 +
===Transfer of m-yCCS into pEX===
 +
 +
====Digestion====
 +
[pEX.RFP] = 82 ng/&mu;l
 +
[pSB1C3.m-yCCS] = 101.2 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|align="center"|[&mu;l]
 +
!width="50"|SOD
 +
!width="50"|pMA.His
 +
|-
 +
|10X FD buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|DNA (1 &mu;g)
 +
|align="center"|12
 +
|align="center"|6
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|13
 +
|align="center"|19
 +
|-
 +
|FD XbaI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|FD PstI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!30
 +
!30
 +
|}
 +
 +
'''Incubation:'''
 +
*37 &deg;C, 10 min
 +
 +
'''Inactivation:'''
 +
*65 &deg;C, 20 min
 +
 +
====Ligation====
 +
 +
{|border="1" cellpadding="1" cellspacing="1"
 +
|&nbsp;
 +
|align="center"|[&mu;l]
 +
|-
 +
|5X Rapid ligation buf.
 +
|width="40" align="center"|4
 +
|-
 +
|Vector DNA (pEX)
 +
|align="center"|1.5
 +
|-
 +
|Insert DNA (m-yCCS)
 +
|align="center"|9
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|4.5
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20
 +
|}
 +
 +
'''Incubation:'''
 +
*22 &deg;C, 10 min
 +
 +
====Transformation====
 +
Quick-transformation (Top10)
 +
*2 &mu;l
 +
*Amp 100
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== MITF-M, starting over... ===
 +
 +
==== Colony PCR ====
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
| rowspan="10" width="150" |
 +
! Primers
 +
| rowspan="10" width="150" |
 +
! colspan="2" | Conditions
 +
| rowspan="3" |
 +
|-
 +
| sH<sub>2</sub>O
 +
| 22.5
 +
| MITF_FB_F_18aug
 +
! time
 +
! &deg;C
 +
|-
 +
| F primer
 +
| 1
 +
| MITF_FB_R_18aug
 +
| 2m
 +
| 95
 +
|-
 +
| R Primer
 +
| 1
 +
| rowspan="7" |
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| DNA
 +
| 0.5
 +
| 30s
 +
| 55
 +
| > 5 cycles
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
| 1m40s
 +
| 72
 +
| )
 +
|-
 +
| rowspan="4" colspan="2" |
 +
| 30s
 +
| 95
 +
| \ 25 cycles
 +
|-
 +
| 2m10s
 +
| 72
 +
| /
 +
|-
 +
| 10m
 +
| 72
 +
|-
 +
| oo
 +
| 10
 +
|}
 +
 +
 +
==== Gel ====
 +
 +
[[Image:2010-09-01_MITF-M_+_FB.jpg|100px|thumb|left|]]
 +
{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| 1kb ladder
 +
|-
 +
| 2
 +
| MITF-M + FB
 +
|}
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
==== Digestion ====
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
| (µl)
 +
| rowspan="7" width="100" |
 +
! colspan="2" | Conditions
 +
|-
 +
| MITF/pSB1C3
 +
| 22
 +
| 20
 +
! Time
 +
! &deg;C
 +
|-
 +
| s<sub>2</sub>O
 +
| 10
 +
| 5
 +
| 30m
 +
| 37
 +
|-
 +
| 10x buffer
 +
| 4
 +
| 3
 +
| 20m
 +
| 65
 +
|-
 +
| EcoRI
 +
| 2
 +
| 1
 +
| oo
 +
| 10
 +
|-
 +
| SpeI
 +
| 2
 +
| 1
 +
| rowspan="2" colspan"2" |
 +
|-
 +
| align="right" | tot
 +
| 40µl
 +
| 30µl
 +
|}
 +
 +
 +
 +
==== Ligation ====
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
| rowspan="7" width="100" |
 +
! colspan="2" | Conditions
 +
|-
 +
| pSB1C3
 +
| 3
 +
! Time
 +
! &deg;C
 +
|-
 +
| MITF-M
 +
| 10
 +
| 10m
 +
| 22
 +
|-
 +
| 5x buffer
 +
| 4
 +
| oo
 +
| 4
 +
|-
 +
| T4 ligase
 +
| 1
 +
| rowspan="3" colspan"2" |
 +
|-
 +
| s<sub>2</sub>O
 +
| 2
 +
|-
 +
| align="right" | tot
 +
| 20µl
 +
|}
 +
 +
{{Stockholm/Footer}}

Latest revision as of 10:58, 26 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

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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/

Retrieved from "http://2010.igem.org/Team:Stockholm/1_September_2010"