Team:Stockholm/30 August 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
 +
==Andreas==
==Andreas==
 +
===Cloning of SOD into pMA.His===
 +
====Transformation results====
 +
''From 28 28/8''
 +
Good colony yield. Four colonies (SH1-SH4) picked for colony PCR.
 +
 +
====Colony PCR====
 +
*SH1-SH4: pMA.SOD⋅His
 +
*PC: Positive control; pMA.His
 +
*NC: Negative control; blank
 +
Procedures according to standard colony PCR protocol. Elongation time 1:00.
 +
 +
====Gel verification====
 +
[[image:ColPCR_pMA.SOD*His_30aug.png|200px|thumb|right|'''Gel verification of SOD cloning into pMA.His.'''<br />3 &mu;l &lambda;, 4 &mu;l sample.<br />&lambda;=O'GeneRuler 1 kb DNA ladder.]]
 +
1 % agarose, 100 V
 +
 +
Expected bands:
 +
*'''pMA.SOD&sdot;His:''' 831 bp
 +
*'''pMA.His:''' 348 bp
 +
 +
''Results''<br />
 +
Well corresponding bands indicating successful insertion of SOD into the vector.
 +
 +
====ON cultures====
 +
SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 &deg;C, 225 rpm.
 +
 +
===N-CPP extraction===
 +
====Gel extraction====
 +
''From 28/8 samples''
 +
Purification using the E.Z.N.A. Gel Extraction kit. Elution in 30 &mu;l dH<sub>2</sub>O; double elution.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|+ align="bottom"|&dagger; ''Phenolate contaminated sample? High absorbtion at 230 nm'' [http://en.wikipedia.org/wiki/Nucleic_acids_analysis#Other_common_contaminants]
 +
!colspan="3"|DNA concentrations
 +
|-
 +
!Sample
 +
!Conc. [ng/&mu;l]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|Tra10
 +
|align="center"|13.56
 +
|align="center"|1.86
 +
|-
 +
|TAT
 +
|align="center"|1.736
 +
|align="center"|1.20
 +
|-
 +
|LMWP &dagger;
 +
|align="center"|2.523
 +
|align="center"|2.69
 +
|}
 +
 +
====Gel verification====
 +
[[image:Gelver_extr_CPP_30aug.png|200px|thumb|right|'''Gel verification of extracted N-CPPs.'''<br />3 &mu;l &lambda;; 2 &mu;l sample.<br />&lambda;=GeneRuler 50 bp DNA ladder]]
 +
Ran a gel to verify that the presence and size of our extracted DNA fragments.
 +
 +
1 % agarose, 100 V
 +
 +
'''Results'''<br />
 +
Weak band for Tra10, no bands for TAT and LMWP. Proceeded to cloning anyway.
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
A last-minute decision was made to also make a bulk cloning of all three N-CPPs by digesting directly from the N-CPP cluster vector.
 +
====Digestion====
 +
[N-CPP plasmid]=672 ng/&mu;l (28/8)
 +
{|border="1" cellpadding="1" cellspacing="1"
 +
|align="center"|[ng/&mu;l]
 +
!Tra10
 +
!TAT
 +
!LMWP
 +
!N-CPP
 +
|-
 +
|10X FD buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|8
 +
|align="center"|23
 +
|-
 +
|FD XbaI
 +
|align="center"|0.5
 +
|align="center"|0.5
 +
|align="center"|0.5
 +
|align="center"|0.5
 +
|-
 +
|FD AgeI
 +
|align="center"|0.5
 +
|align="center"|0.5
 +
|align="center"|0.5
 +
|align="center"|0.5
 +
|-
 +
|DNA
 +
|align="center"|23
 +
|align="center"|23
 +
|align="center"|18
 +
|align="center"|3
 +
|-
 +
|&nbsp;
 +
!30
 +
!30
 +
!30
 +
!30
 +
|}
 +
 +
Incubation: 37 &deg;C, 30 min<br />
 +
Inactivation: 80 &deg;C, 10 min
 +
 +
=====Dephosphorylation=====
 +
Treated the N-CPP sample with FastAP alkaline phosphatase to prevent multiple insertions (3, 5, etc...) into target vector.
 +
*3 &mu;l FastAP
 +
**Incubation: 37 &deg;C, 10 min
 +
*Inactivation at 75 &deg;C, 5 min
 +
 +
====Ligation====
 +
[Dig. pSB1X3 X+A EXTR]=13.72 ng/&mu;l (digested and extracted vector from 9/8)
 +
[Dig. N-CPP X+A]=60 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|align="center"|[ng/&mu;l]
 +
!N-CPP
 +
!Tra10
 +
!TAT
 +
!LMWP
 +
|-
 +
|Vector DNA
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|Insert DNA
 +
|align="center"|9
 +
|align="center"|12
 +
|align="center"|12
 +
|align="center"|12
 +
|-
 +
|5X Rapid Lig. buf.
 +
|align="center"|4
 +
|align="center"|4
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|3
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|-
 +
|T4 DNA Ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20
 +
!20
 +
!20
 +
!20
 +
|}
 +
 +
====Transformation====
 +
Standard transformation protocol.
 +
*2 &mu;l ligation mix
 +
*Cm 25
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== MITF-M ===
 +
 +
==== Colony PCR ====
 +
 +
 +
*Made by Andreas --> ~280bp bands, empty vector? should not be possible...
 +
 +
*Try again, more colonies
 +
 +
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
| X8
 +
| rowspan="8" width="150" |
 +
! Primers
 +
| rowspan="8" width="150" |
 +
! colspan="2" | Conditions
 +
| rowspan="3" |
 +
|-
 +
| sH<sub>2</sub>O
 +
| 22.5
 +
| 180
 +
| pSB1_VF2
 +
! time
 +
! &deg;C
 +
|-
 +
| F primer
 +
| 1
 +
| 8
 +
| pSB1_VR
 +
| 2m
 +
| 94
 +
|-
 +
| R primer
 +
| 1
 +
| 8
 +
| rowspan="5" |
 +
| 30s
 +
| 94
 +
| )
 +
|-
 +
| DNA
 +
| 0.5
 +
| 8x0.5
 +
| 30s
 +
| 50
 +
| > 30 cycles
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
|
 +
| 2m40s
 +
| 72
 +
| )
 +
|-
 +
| rowspan="2" colspan="3" |
 +
| 10m
 +
| 72
 +
| rowspan="2" |
 +
|-
 +
| oo
 +
| 10
 +
|}
 +
 +
 +
 +
==== Gel ====
 +
 +
{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| pSB1C3.MITF-M 1
 +
|-
 +
| 3
 +
| pSB1C3.MITF-M 2
 +
|-
 +
| 4
 +
| pSB1C3.MITF-M 3
 +
|-
 +
| 5
 +
| pSB1C3.MITF-M 4
 +
|-
 +
| 6
 +
| pSB1C3.MITF-M 5
 +
|-
 +
| 7
 +
| pSB1C3.MITF-M 6
 +
|-
 +
| 8
 +
| pSB1C3.MITF-M 7
 +
|-
 +
| 9
 +
| blank
 +
|}
 +
 +
 +
*No product, trying another 7 colonies over night...
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:03, 26 October 2010

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Contents

Andreas

Cloning of SOD into pMA.His

Transformation results

From 28 28/8 Good colony yield. Four colonies (SH1-SH4) picked for colony PCR.

Colony PCR

  • SH1-SH4: pMA.SOD⋅His
  • PC: Positive control; pMA.His
  • NC: Negative control; blank

Procedures according to standard colony PCR protocol. Elongation time 1:00.

Gel verification

Gel verification of SOD cloning into pMA.His.
3 μl λ, 4 μl sample.
λ=O'GeneRuler 1 kb DNA ladder.

1 % agarose, 100 V

Expected bands:

  • pMA.SOD⋅His: 831 bp
  • pMA.His: 348 bp

Results
Well corresponding bands indicating successful insertion of SOD into the vector.

ON cultures

SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm.

N-CPP extraction

Gel extraction

From 28/8 samples Purification using the E.Z.N.A. Gel Extraction kit. Elution in 30 μl dH2O; double elution.

† Phenolate contaminated sample? High absorbtion at 230 nm [http://en.wikipedia.org/wiki/Nucleic_acids_analysis#Other_common_contaminants]
DNA concentrations
Sample Conc. [ng/μl] A260/A280
Tra10 13.56 1.86
TAT 1.736 1.20
LMWP † 2.523 2.69

Gel verification

Gel verification of extracted N-CPPs.
3 μl λ; 2 μl sample.
λ=GeneRuler 50 bp DNA ladder

Ran a gel to verify that the presence and size of our extracted DNA fragments.

1 % agarose, 100 V

Results
Weak band for Tra10, no bands for TAT and LMWP. Proceeded to cloning anyway.

Cloning of N-CPPs into pSB1C3

A last-minute decision was made to also make a bulk cloning of all three N-CPPs by digesting directly from the N-CPP cluster vector.

Digestion

[N-CPP plasmid]=672 ng/μl (28/8)

[ng/μl] Tra10 TAT LMWP N-CPP
10X FD buffer 3 3 3 3
dH2O 3 3 8 23
FD XbaI 0.5 0.5 0.5 0.5
FD AgeI 0.5 0.5 0.5 0.5
DNA 23 23 18 3
  30 30 30 30

Incubation: 37 °C, 30 min
Inactivation: 80 °C, 10 min

Dephosphorylation

Treated the N-CPP sample with FastAP alkaline phosphatase to prevent multiple insertions (3, 5, etc...) into target vector.

  • 3 μl FastAP
    • Incubation: 37 °C, 10 min
  • Inactivation at 75 °C, 5 min

Ligation

[Dig. pSB1X3 X+A EXTR]=13.72 ng/μl (digested and extracted vector from 9/8) [Dig. N-CPP X+A]=60 ng/μl

[ng/μl] N-CPP Tra10 TAT LMWP
Vector DNA 3 3 3 3
Insert DNA 9 12 12 12
5X Rapid Lig. buf. 4 4 4 4
dH2O 3 0 0 0
T4 DNA Ligase 1 1 1 1
  20 20 20 20

Transformation

Standard transformation protocol.

  • 2 μl ligation mix
  • Cm 25



Mimmi

MITF-M

Colony PCR

  • Made by Andreas --> ~280bp bands, empty vector? should not be possible...
  • Try again, more colonies


Mix (µl) X8 Primers Conditions
sH2O 22.5 180 pSB1_VF2 time °C
F primer 1 8 pSB1_VR 2m 94
R primer 1 8 30s 94 )
DNA 0.5 8x0.5 30s 50 > 30 cycles
tot 25µl 2m40s 72 )
10m 72
oo 10


Gel

well sample
1 ladder
2 pSB1C3.MITF-M 1
3 pSB1C3.MITF-M 2
4 pSB1C3.MITF-M 3
5 pSB1C3.MITF-M 4
6 pSB1C3.MITF-M 5
7 pSB1C3.MITF-M 6
8 pSB1C3.MITF-M 7
9 blank


  • No product, trying another 7 colonies over night...





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

Retrieved from "http://2010.igem.org/Team:Stockholm/30_August_2010"