Team:Stockholm/30 August 2010

From 2010.igem.org

(Difference between revisions)
(→Andreas)
(→Andreas)
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====ON cultures====
====ON cultures====
SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm.
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 &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

Revision as of 12:21, 31 August 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
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