Team:Stockholm/24 September 2010

From 2010.igem.org

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==Andreas==
==Andreas==
 +
===Plasmid prep===
 +
''From 23/9 ON cultures''
 +
 +
Omega E.Z.N.A. Plasmid Miniprep kit I.
 +
:New plasmid prep buffers
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|+align="bottom"|† = ''unsure value due to bad blank sample.''
 +
|-
 +
!Sample
 +
!width="60"|Conc [ng/μl]
 +
!width="60"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1A2.RBS.yCCS 3
 +
|align="center"|195.5
 +
|align="center"|1.81
 +
|-
 +
|pSB1A2.RBS.yCCS 4
 +
|align="center"|165.7
 +
|align="center"|1.85
 +
|-
 +
|pEX.N-TAT&sdot;SOD&sdot;His 3&dagger;
 +
|align="center"|60.00
 +
|align="center"|1.83
 +
|-
 +
|pEX.N-TAT&sdot;SOD&sdot;His 4&dagger;
 +
|align="center"|90.00
 +
|align="center"|1.73
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 4
 +
|align="center"|130.5
 +
|align="center"|1.84
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 5&dagger;
 +
|align="center"|60.00
 +
|align="center"|1.88
 +
|-
 +
|pSB1K3.N-Tra10&sdot;SOD&sdot;His 5&dagger;
 +
|align="center"|90.00
 +
|align="center"|1.93
 +
|-
 +
|pSB1K3.N-LMWP&sdot;SOD&sdot;His 1
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|align="center"|258.2
 +
|align="center"|1.87
 +
|-
 +
|pSB1C3.N-LMWP&sdot;SOD&sdot;His 1
 +
|align="center"|155.4
 +
|align="center"|1.82
 +
|-
 +
|pSB1C3.N-LMWP&sdot;SOD&sdot;His 4
 +
|align="center"|331.9
 +
|align="center"|1.80
 +
|}
 +
 +
===Cloning and assembly===
 +
 +
====Digestions====
 +
 +
[pEX.RFP] = 44.0 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
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!width="70"|pA.RBS. yCCS (3) X+P
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!width="70"|pEX.N-TAT. SH (4) X+P
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!width="70"|pK.N-TAT. SH (4) S+P
 +
!width="70"|pK.N-Tra10. SH (5) X+P
 +
!width="70"|pK.N-Tra10. SH (5) S+P
 +
!width="70"|pC.N-LMWP. SH (4) X+P
 +
!width="70"|pC.N-LMWP. SH (4) S+P
 +
!width="70"|pEX.RFP X+P
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|3
 +
|-
 +
|DNA (1 &mu;g)
 +
|align="center"|5
 +
|align="center"|12.5
 +
|align="center"|8
 +
|align="center"|12.5
 +
|align="center"|12.5
 +
|align="center"|4
 +
|align="center"|4
 +
|align="center"|22
 +
|-
 +
|dH<sub>2</sub>O
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|align="center"|11
 +
|align="center"|3.5
 +
|align="center"|7
 +
|align="center"|3.5
 +
|align="center"|3.5
 +
|align="center"|12
 +
|align="center"|12
 +
|align="center"|3
 +
|-
 +
|FD XbaI
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|-
 +
|FD PstI
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
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|FD SpeI
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!30 &mu;l
 +
|}
 +
*Incubation: 37 &deg;C, 1 h
 +
 +
====Gel verification====
 +
[[image:Gelver_digestions_24sep.png|200px|thumb|right|'''Gel verification of sample digestions.'''<br />4 &mu;l &lambda;; 3 &mu;l sample.<br />1 kb &lambda; = O'GeneRuler 1 kb DNA ladder. 50 bp &lambda; = GeneRuler 50 bp DNA ladder.]]
 +
''Due to the risk of pSB1A2.RBS.yCCS and pEX.N-TAT&sdot;SOD&sdot;His having previously been mixed up, a gel was run on the digested samples (after 15 min incubation) to verify the excised insert size. Remaining samples were also run on the gel to verify digestion.
 +
 +
1 % agarose, 120 V
 +
 +
'''Expected bands'''
 +
#'''pSB1A2.RBS.yCCS X+P:''' 806 bp, 2061 bp
 +
#'''pEX.N-TAT&sdot;SOD&sdot;His X+P:''' 558 bp, 4453 bp
 +
#'''pSB1K3.N-TAT&sdot;SOD&sdot;His S+P:''' &asymp;2730 bp
 +
#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His X+P:''' 588 bp, 2188 bp
 +
#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His S+P:''' &asymp;2760 bp
 +
#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His X+P:''' 567 bp, 2054 bp
 +
#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His S+P:''' &asymp;2610 bp
 +
#'''pEX.RFP X+P:''' 1095 bp, 4453 bp
 +
 +
'''Results'''<br />
 +
Seemingly correct bands for samples 1, 3, 4, 5 and 8. More unsure results for 6 and 7, while 2 seems to contain more than one insert, or has been digested at several places.
 +
 +
==Nina==
 +
 +
===Overnight culture===
 +
 +
I inoculated protein A#5_TAT, _LMWP & _Tra10 all from colony #1 on each dish.
 +
 +
12 ml LB + 24 ul chloramphenicol.
 +
 +
===Concentration measurement===
 +
 +
*Protein A#5_CPP_TAT_C cons: 95ng/ul 95/10 = 9.5ng/ul λ260 0.019 λ280 0.009 λ315 -0.002
 +
 +
*Vector with CPP_TAT_C conc: 95 ng/ul
 +
 +
95ng/ul * Volume = 25ng/ul * 5 ul
 +
 +
Volume = 1.3 ul sample and fill up to 5 ml with H2O.
 +
 +
*IgG N+P cons: 30ng/ul 30/2.5 = 12ng/ul λ260 0.007 λ280 0.006 λ315 0.000
 +
 +
*IgG A+E cons: 40ng/ul 40/2.5 = 16ng/ul λ260 0.007 λ280 0.007 λ315 0.001
 +
 +
===Ligation===
 +
 +
*Vector CPP_TAT_C 1 ul
 +
*Protein A gene 21 ul
 +
*Quick ligase 1 ul
 +
*Quicke ligase buffer 2X 23 ul
 +
 +
 +
 +
 +
*Vector LMWP, TAT & Tra10 0.5 ul each
 +
*gene IgG (N+P) 7.5 ul each
 +
*Quick ligation 1 ul
 +
*Quick ligation buffer 2X 9 ul each
 +
 +
 +
 +
 +
 +
*Vector CPP_TAT_C 1 ul
 +
*gene IgG (A+E) 17 ul
 +
*Quick ligation 1 ul
 +
*Quick ligation buffer 2X 19 ul
 +
 +
===Transformation===
 +
 +
I transformed the ligations with 10 ul in 100 ul Top 10 cloning cells.
 +
 +
===Digestion===
 +
 +
*H2O 15 ul
 +
*Fastdigest buffer 10X 2 ul
 +
*DNA 2 ul
 +
*Restriction enzyme NgoMIV 1 ul
 +
*Restriction enzyme PstI 1 ul (Add after 1.5 h incubation in 37 °C and incubate in 30 min)
 +
 +
Inactivate in 80 °C for 30 min.
 +
 +
Added CIAP 1 ul and incubate 1 h in 37 °C.
 +
 +
 
 +
 +
==Johan==
 +
 +
===Gel purification===
 +
 +
A gel was run with pMA (vector with histag) cut before and after his, and bFGF cut before and after
 +
 +
A gel purification was then performed on all samples.
 +
 +
Abs:
 +
 +
bFGF-his: 14 ng/µl
 +
his-bFGF: 18 ng/µl
 +
pMA before: 22 ng/µl
 +
pMA after: 30 ng /µl
 +
 +
===Ligation===
 +
 +
bFGF after & pMA before
 +
 +
bFGF before & pMA after
 +
 +
5 µl insert
 +
 +
3 µl vector
 +
 +
1 µl T4 ligase
 +
 +
2 µl 10x buffer
 +
 +
9 µl H2O
 +
 +
===Transformation===
 +
 +
3 µl of all constructs was transformed into top10 cells.
 +
 +
{{Stockholm/Footer}}

Latest revision as of 22:13, 27 October 2010

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Contents

Andreas

Plasmid prep

From 23/9 ON cultures

Omega E.Z.N.A. Plasmid Miniprep kit I.

New plasmid prep buffers
DNA concentration
† = unsure value due to bad blank sample.
Sample Conc [ng/μl] A260/A280
pSB1A2.RBS.yCCS 3 195.5 1.81
pSB1A2.RBS.yCCS 4 165.7 1.85
pEX.N-TAT⋅SOD⋅His 3† 60.00 1.83
pEX.N-TAT⋅SOD⋅His 4† 90.00 1.73
pSB1K3.N-TAT⋅SOD⋅His 4 130.5 1.84
pSB1K3.N-TAT⋅SOD⋅His 5† 60.00 1.88
pSB1K3.N-Tra10⋅SOD⋅His 5† 90.00 1.93
pSB1K3.N-LMWP⋅SOD⋅His 1 258.2 1.87
pSB1C3.N-LMWP⋅SOD⋅His 1 155.4 1.82
pSB1C3.N-LMWP⋅SOD⋅His 4 331.9 1.80

Cloning and assembly

Digestions

[pEX.RFP] = 44.0 ng/μl

  pA.RBS. yCCS (3) X+P pEX.N-TAT. SH (4) X+P pK.N-TAT. SH (4) S+P pK.N-Tra10. SH (5) X+P pK.N-Tra10. SH (5) S+P pC.N-LMWP. SH (4) X+P pC.N-LMWP. SH (4) S+P pEX.RFP X+P
10X FastDigest buffer 2 2 2 2 2 2 2 3
DNA (1 μg) 5 12.5 8 12.5 12.5 4 4 22
dH2O 11 3.5 7 3.5 3.5 12 12 3
FD XbaI 1 1 0 1 0 1 0 1
FD PstI 1 1 1 1 1 1 1 1
FD SpeI 0 0 1 0 1 0 1 0
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 30 μl
  • Incubation: 37 °C, 1 h

Gel verification

Gel verification of sample digestions.
4 μl λ; 3 μl sample.
1 kb λ = O'GeneRuler 1 kb DNA ladder. 50 bp λ = GeneRuler 50 bp DNA ladder.

Due to the risk of pSB1A2.RBS.yCCS and pEX.N-TAT⋅SOD⋅His having previously been mixed up, a gel was run on the digested samples (after 15 min incubation) to verify the excised insert size. Remaining samples were also run on the gel to verify digestion.

1 % agarose, 120 V

Expected bands

  1. pSB1A2.RBS.yCCS X+P: 806 bp, 2061 bp
  2. pEX.N-TAT⋅SOD⋅His X+P: 558 bp, 4453 bp
  3. pSB1K3.N-TAT⋅SOD⋅His S+P: ≈2730 bp
  4. pSB1K3.N-Tra10⋅SOD⋅His X+P: 588 bp, 2188 bp
  5. pSB1K3.N-Tra10⋅SOD⋅His S+P: ≈2760 bp
  6. pSB1C3.N-LMWP⋅SOD⋅His X+P: 567 bp, 2054 bp
  7. pSB1C3.N-LMWP⋅SOD⋅His S+P: ≈2610 bp
  8. pEX.RFP X+P: 1095 bp, 4453 bp

Results
Seemingly correct bands for samples 1, 3, 4, 5 and 8. More unsure results for 6 and 7, while 2 seems to contain more than one insert, or has been digested at several places.

Nina

Overnight culture

I inoculated protein A#5_TAT, _LMWP & _Tra10 all from colony #1 on each dish.

12 ml LB + 24 ul chloramphenicol.

Concentration measurement

  • Protein A#5_CPP_TAT_C cons: 95ng/ul 95/10 = 9.5ng/ul λ260 0.019 λ280 0.009 λ315 -0.002
  • Vector with CPP_TAT_C conc: 95 ng/ul

95ng/ul * Volume = 25ng/ul * 5 ul

Volume = 1.3 ul sample and fill up to 5 ml with H2O.

  • IgG N+P cons: 30ng/ul 30/2.5 = 12ng/ul λ260 0.007 λ280 0.006 λ315 0.000
  • IgG A+E cons: 40ng/ul 40/2.5 = 16ng/ul λ260 0.007 λ280 0.007 λ315 0.001

Ligation

  • Vector CPP_TAT_C 1 ul
  • Protein A gene 21 ul
  • Quick ligase 1 ul
  • Quicke ligase buffer 2X 23 ul



  • Vector LMWP, TAT & Tra10 0.5 ul each
  • gene IgG (N+P) 7.5 ul each
  • Quick ligation 1 ul
  • Quick ligation buffer 2X 9 ul each



  • Vector CPP_TAT_C 1 ul
  • gene IgG (A+E) 17 ul
  • Quick ligation 1 ul
  • Quick ligation buffer 2X 19 ul

Transformation

I transformed the ligations with 10 ul in 100 ul Top 10 cloning cells.

Digestion

  • H2O 15 ul
  • Fastdigest buffer 10X 2 ul
  • DNA 2 ul
  • Restriction enzyme NgoMIV 1 ul
  • Restriction enzyme PstI 1 ul (Add after 1.5 h incubation in 37 °C and incubate in 30 min)

Inactivate in 80 °C for 30 min.

Added CIAP 1 ul and incubate 1 h in 37 °C.


Johan

Gel purification

A gel was run with pMA (vector with histag) cut before and after his, and bFGF cut before and after

A gel purification was then performed on all samples.

Abs:

bFGF-his: 14 ng/µl his-bFGF: 18 ng/µl pMA before: 22 ng/µl pMA after: 30 ng /µl

Ligation

bFGF after & pMA before

bFGF before & pMA after

5 µl insert

3 µl vector

1 µl T4 ligase

2 µl 10x buffer

9 µl H2O

Transformation

3 µl of all constructs was transformed into top10 cells.





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

Retrieved from "http://2010.igem.org/Team:Stockholm/24_September_2010"