Team:Stockholm/7 September 2010

From 2010.igem.org

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(→Andreas)
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**5 ml LB + Cm 25
**5 ml LB + Cm 25
**37 °C, 200 rpm ON
**37 °C, 200 rpm ON
 +
 +
====PCR product digestion====
 +
''From 6/9 PCR''
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|10X Buffer Tango
 +
|align="center" width="50"|5
 +
|-
 +
|PCR DNA
 +
|align="center"|40
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|-
 +
|AgeI
 +
|align="center"|2
 +
|-
 +
|XbaI
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!50 &mu;l
 +
|}
 +
 +
*Incubation: 37 &deg;C, 3 h
 +
*Inactivation: 80 &deg;C, 20 min
 +
 +
====Gel verification====
 +
[[image:Gelver_N-CPP_dig_7sep.png|200px|thumb|right|'''Gel verification of N-CPP digestions.'''3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = GeneRuler 50 bp DNA ladder.]]
 +
1.5 % agarose, 90 V
 +
 +
'''Expected bands'''
 +
*Tra10: 182 bp
 +
*TAT: 91 bp
 +
*LMWP: 94 bp
 +
 +
'''Results'''<br />
 +
Very irregular band sizes in the "lower" region, making no sense. Sizes not at all corresponding to what was expected.<br />
 +
Higher up very regular bands. This is even more puzzling, since different primer pairs were used for each PCR reaction. Although concentration of plasmid should be too low, the bands seen may be undigested and digested N-CPP plasmid. A too large plasmid concentration may in turn be the cause of the unspecific bands seen lower down in the gel.
===Transfer of m-yCCS into pEX===
===Transfer of m-yCCS into pEX===
Line 73: Line 112:
**5 ml LB + Amp 100
**5 ml LB + Amp 100
**30 &deg;C
**30 &deg;C
 +
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== SOD.his / his.SOD ===
 +
 +
==== col-PCR ====
 +
 +
{|
 +
! mix
 +
| (µl)
 +
| x9 x2
 +
| rowspan="9" width="100" |
 +
! primers
 +
| rowspan="9" width="100" |
 +
! colspan="2" | conditions
 +
| rowspan="3" |
 +
|-
 +
| sH<sub>2</sub>O
 +
| 13.5
 +
| 121.5
 +
| pSB_VF2
 +
! time
 +
! &deg;C
 +
|-
 +
| dNTP
 +
| 2
 +
| 18
 +
| pSB_VR
 +
| 2m
 +
| 95
 +
|-
 +
| F primer
 +
| 0.8
 +
| 7.2
 +
| &
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| R primer
 +
| 0.8
 +
| 7.2
 +
| pEX_VF
 +
| 30s
 +
| 55
 +
| > 30 cycles
 +
|-
 +
| buffer
 +
| 2
 +
| 18
 +
| pEX_VR
 +
| 1m45s
 +
| 72
 +
| )
 +
|-
 +
| polymerase
 +
| 0.4
 +
| 3.6
 +
| rowspan="3" |
 +
| 10m
 +
| 72
 +
| rowspan="3" |
 +
|-
 +
| DNA
 +
| 0.5
 +
|
 +
| OO
 +
| 10
 +
|-
 +
| align="right" | tot
 +
| 20µl
 +
| 180µlx2
 +
|}
 +
 +
 +
 +
==== gel ====
 +
 +
{|
 +
! well
 +
! width="150" | sample
 +
! well
 +
! width="150" | sample
 +
|-
 +
| 1
 +
| ladder
 +
| 12
 +
| ladder
 +
|-
 +
| 2
 +
| pSB1C3.SOD.his 1
 +
| 13
 +
| pEX.SOD.his 1
 +
|-
 +
| 3
 +
| pSB1C3.SOD.his 2
 +
| 14
 +
| pEX.SOD.his 2
 +
|-
 +
| 4
 +
| pSB1C3.SOD.his 3
 +
| 15
 +
| pEX.SOD.his 3
 +
|-
 +
| 5
 +
| pSB1C3.SOD.his 4
 +
| 16
 +
| pEX.SOD.his 4
 +
|-
 +
| 6
 +
| pSB1C3.his.SOD 1
 +
| 17
 +
| pEX.his.SOD 1
 +
|-
 +
| 7
 +
| pSB1C3.his.SOD 2
 +
| 18
 +
| pEX.his.SOD 2
 +
|-
 +
| 8
 +
| pSB1C3.his.SOD 3
 +
| 19
 +
| pEX.his.SOD 3
 +
|-
 +
| 9
 +
| pSB1C3.his.SOD 4
 +
| 20
 +
| pEX.his.SOD 4
 +
|-
 +
| 10
 +
| positive control
 +
| 21
 +
| blank
 +
|}
 +
 +
 +
* Nothing...
 +
 +
=== MITF-M ===
 +
 +
==== Digestion ====
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
|
 +
| (µl)
 +
| rowspan="7" width="100" |
 +
! colspan="2" | Conditions
 +
|-
 +
| DNA
 +
| 40
 +
|
 +
| 10
 +
! Time
 +
! &deg;C
 +
|-
 +
| 10x buffer
 +
| 5
 +
|
 +
| 5
 +
| 30m
 +
| 37
 +
|-
 +
| sH<sub>2</sub>O
 +
| 3
 +
|
 +
| 4
 +
| 20m
 +
| 65
 +
|-
 +
| EcoRI
 +
| 1
 +
| PstI
 +
| 1
 +
| oo
 +
| 10
 +
|-
 +
| SpeI
 +
| 1
 +
|
 +
| 0
 +
| colspan="2" rowspan="2" |
 +
|-
 +
| align="right" | tot
 +
| 50µl
 +
|
 +
| 20µl
 +
|}
 +
 +
 +
==== Gel ====
 +
 +
[[Image:2010-09-07_MITF_dig&lig.jpg|200px|thumb|left|]]
 +
{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| MITF-M
 +
|-
 +
| 3
 +
| MITF-M
 +
|-
 +
| 4
 +
| MITF cut E+S
 +
|-
 +
| 5
 +
| MITF cut P
 +
|-
 +
| 6
 +
| pSB1C3 cut E+S
 +
|-
 +
| 7
 +
| pSB1C3.MITF lig
 +
|-
 +
| 8
 +
| pSB1C3.MITF lig
 +
|}
 +
 +
==== Ligation (nr3) ====
 +
 +
{|
 +
! mix
 +
| (µl)
 +
| rowspan="7" width="70" |
 +
| [pSB1C3]~100ng/µl
 +
| rowspan="7" width="70" |
 +
! colspan="2" | conditions
 +
|-
 +
| pSB1C3
 +
| 2
 +
| rowspan="6" |
 +
! time
 +
! &deg;C
 +
|-
 +
| MITF-M
 +
| 13
 +
| 10m
 +
| 22
 +
|-
 +
| 5xbuffer
 +
| 4
 +
| colspan="2" rowspan="4" |
 +
|-
 +
| T4 ligase
 +
| 1
 +
|-
 +
| sH<sub>2</sub>O
 +
| 0
 +
|-
 +
| align="right" | tot
 +
| 20µl
 +
|}
 +
 +
 +
==== Transformation ====
 +
 +
*Follow original protocol
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:00, 26 October 2010

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

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Contents

Andreas

Cloning of N-CPPs into pSB1C3

Transformation results

From 6/9 transformations

Good colony yields on both plates, 1 and 2*.

Colony PCR

4 colonies picked from each plate for verification by colony PCR.

  • 1, 2, 3, 4, 5*, 6*, 7*, 8*
  • PC: pSB1C3.RFP
PCR tubes
dH2O 16.22
DreamTaq buffer 2
dNTP, 10 mM 0.4
Fwd primer (VF2) 0.4
Rev primer (VR) 0.4
Cell suspension 0.5
DreamTaq pol. 0.08
  20 μl

Standard colony PCR settings

  • Elongation time: 0:45

Gel verification

Colony PCR gel verification of pSB1C3.N-CPPs
3 μl λ; 6 μl sample.
λ = GeneRuler 50 bp DNA ladder.

1.5 % agarose, 90 V.

Expected bands

  • pSB1C3.Tra10: 389 bp
  • pSB1C3.TAT: 359 bp
  • pSB1C3.LMWP: 368 bp

Results
Relevant bands in clones 3, 4, 6, 7 & 8. Slightly different in size, which could indicate presence of all three N-CPPs. All clones selected for sequencing.

ON cultures

For plasmid prep

  • pSB1C3.N-CPP: pC.NCPP 3, 4, 6*, 7* and 8*
    • 5 ml LB + Cm 25
    • 37 °C, 200 rpm ON

PCR product digestion

From 6/9 PCR

10X Buffer Tango 5
PCR DNA 40
dH2O 0
AgeI 2
XbaI 1
  50 μl
  • Incubation: 37 °C, 3 h
  • Inactivation: 80 °C, 20 min

Gel verification

Gel verification of N-CPP digestions.3 μl λ; 5 μl sample.
λ = GeneRuler 50 bp DNA ladder.

1.5 % agarose, 90 V

Expected bands

  • Tra10: 182 bp
  • TAT: 91 bp
  • LMWP: 94 bp

Results
Very irregular band sizes in the "lower" region, making no sense. Sizes not at all corresponding to what was expected.
Higher up very regular bands. This is even more puzzling, since different primer pairs were used for each PCR reaction. Although concentration of plasmid should be too low, the bands seen may be undigested and digested N-CPP plasmid. A too large plasmid concentration may in turn be the cause of the unspecific bands seen lower down in the gel.

Transfer of m-yCCS into pEX

ON cultures

Set ON cultures of pEX.yCCS for plasmid prep and glycerol stocks. From 3/9 colonies.

  • pEX.yCCS 5 and 8
    • 5 ml LB + Amp 100
    • 37 °C, 200 rpm ON
  • pEX.yCCS 5 and 8
    • 5 ml LB + Amp 100
    • 30 °C




Mimmi

SOD.his / his.SOD

col-PCR

mix (µl) x9 x2 primers conditions
sH2O 13.5 121.5 pSB_VF2 time °C
dNTP 2 18 pSB_VR 2m 95
F primer 0.8 7.2 & 30s 95 )
R primer 0.8 7.2 pEX_VF 30s 55 > 30 cycles
buffer 2 18 pEX_VR 1m45s 72 )
polymerase 0.4 3.6 10m 72
DNA 0.5 OO 10
tot 20µl 180µlx2


gel

well sample well sample
1 ladder 12 ladder
2 pSB1C3.SOD.his 1 13 pEX.SOD.his 1
3 pSB1C3.SOD.his 2 14 pEX.SOD.his 2
4 pSB1C3.SOD.his 3 15 pEX.SOD.his 3
5 pSB1C3.SOD.his 4 16 pEX.SOD.his 4
6 pSB1C3.his.SOD 1 17 pEX.his.SOD 1
7 pSB1C3.his.SOD 2 18 pEX.his.SOD 2
8 pSB1C3.his.SOD 3 19 pEX.his.SOD 3
9 pSB1C3.his.SOD 4 20 pEX.his.SOD 4
10 positive control 21 blank


  • Nothing...

MITF-M

Digestion

Mix (µl) (µl) Conditions
DNA 40 10 Time °C
10x buffer 5 5 30m 37
sH2O 3 4 20m 65
EcoRI 1 PstI 1 oo 10
SpeI 1 0
tot 50µl 20µl


Gel

2010-09-07 MITF dig&lig.jpg
well sample
1 ladder
2 MITF-M
3 MITF-M
4 MITF cut E+S
5 MITF cut P
6 pSB1C3 cut E+S
7 pSB1C3.MITF lig
8 pSB1C3.MITF lig

Ligation (nr3)

mix (µl) [pSB1C3]~100ng/µl conditions
pSB1C3 2 time °C
MITF-M 13 10m 22
5xbuffer 4
T4 ligase 1
sH2O 0
tot 20µl


Transformation

  • Follow original protocol





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

Retrieved from "http://2010.igem.org/Team:Stockholm/7_September_2010"