Team:Stockholm/28 August 2010

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(Difference between revisions)
(→Andreas)
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Elution volume: 30 μl (eluted twice to increase DNA yield)
Elution volume: 30 μl (eluted twice to increase DNA yield)
*Pur. Dig. pMA His E+N
*Pur. Dig. pMA His E+N
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 +
===Gel verification===
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[[image:Gelver_DNAextr_28aug.png|200px|right|thumb|'''Gel verification of gel extracted and purified DNA samples.'''<br />3 &mu;l &lambda;; 2 &mu;l sample.<br />1 kb &lambda;=O'GeneRuler 1 kb DNA ladder. 50 bp &lambda;=GeneRuler 50 bp DNA ladder.]]
 +
Since no DNA content was measurable for "Extr. Dig SOD E+A", a gel was run to verify DNA content in the three purified samples.
 +
 +
1 % agarose, 110 V
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 +
Samples:
 +
*SOD: Extr. Dig SOD E+A
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*pMA: Pur. Dig pMA.His E+N
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*CPP: Extr. N-CPP cluster
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 +
Expected bands:
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*SOD: 492 bp
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*pMA: 2427 bp
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*CPP: 379 bp
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====Results====
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*CPP resulted in a clear band of correct size.
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*A very weak band at &asymp;500 bp is visible for SOD, which might be traces of DNA.
 +
*No band visible for pMA.
 +
 +
Nevertheless, proceeded to ligation and cloning, hoping that the DNA is there.
 +
 +
===Cloning of SOD into pMA.His===
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 +
====Ligation====
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{|border="1" cellpadding="1" cellspacing="0"
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|&nbsp;
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![&mu;l]
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|-
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|Extr. Dig SOD E+A
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|align="center"|12
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|-
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|Pur. Dig pMA.His E+N
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|align="center"|3
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|-
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|5X Rapid Ligation buffer
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|align="center"|4
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|-
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|T4 DNA ligase
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|align="center"|1
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|}
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Incubation: 22 &deg;C, 10 min
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 +
====Transformation====
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*3 &mu;l ligation mix. 30 min on ice.
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*30 sec heat shock in 42 &deg;C
 +
*Cells grown on Amp 100 LB agar, 37 &deg;C
 +
 +
===Amplification of N-CPPs from N-CPP cluster===

Revision as of 12:09, 30 August 2010

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Contents

Andreas

Gel extraction

No measurable DNA conc. for "Extr. Dig SOD E+A"
DNA concentrations
Sample Conc [ng/μl] A260/A280
Extr. N-CPP 40.91 1.69
Extr. Dig SOD E+A – –
Pur. Dig pMA.His E+N 36.02 1.80

From 27/8
Purified DNA from excised samples "Extr. Dig. SOD E+A" and "Extr. N-CPP cluster" using the E.Z.N.A. Gel extraction kit; procedures according to provided protocol.

Elution volume: 30 μl (eluted twice to increase DNA yield)

  • Extr. N-CPP
  • Extr. Dig SOD E+A

DNA purification of "Dig. pMA.His (E+N)"

From 27/8
DNA clean-up using the E.Z.N.A. Gel Extraction kit, following procedures for DNA purification.

Elution volume: 30 μl (eluted twice to increase DNA yield)

  • Pur. Dig. pMA His E+N

Gel verification

Gel verification of gel extracted and purified DNA samples.
3 μl λ; 2 μl sample.
1 kb λ=O'GeneRuler 1 kb DNA ladder. 50 bp λ=GeneRuler 50 bp DNA ladder.

Since no DNA content was measurable for "Extr. Dig SOD E+A", a gel was run to verify DNA content in the three purified samples.

1 % agarose, 110 V

Samples:

  • SOD: Extr. Dig SOD E+A
  • pMA: Pur. Dig pMA.His E+N
  • CPP: Extr. N-CPP cluster

Expected bands:

  • SOD: 492 bp
  • pMA: 2427 bp
  • CPP: 379 bp

Results

  • CPP resulted in a clear band of correct size.
  • A very weak band at ≈500 bp is visible for SOD, which might be traces of DNA.
  • No band visible for pMA.

Nevertheless, proceeded to ligation and cloning, hoping that the DNA is there.

Cloning of SOD into pMA.His

Ligation

  [μl]
Extr. Dig SOD E+A 12
Pur. Dig pMA.His E+N 3
5X Rapid Ligation buffer 4
T4 DNA ligase 1

Incubation: 22 °C, 10 min

Transformation

  • 3 μl ligation mix. 30 min on ice.
  • 30 sec heat shock in 42 °C
  • Cells grown on Amp 100 LB agar, 37 °C

Amplification of N-CPPs from N-CPP cluster

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