Team:Stockholm/11 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
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===Removal of insertion in BioBrick suffixes===
 +
====Plasmid prep====
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''From 8/10 stored pellet''
 +
 +
*50 μl elution buffer.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
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!colspan="3"|DNA concentration
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|-
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!Sample
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!width="60"|Conc [ng/μl]
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!width="60"|A<sub>260</sub>/A<sub>280</sub>
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|-
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|pSB1C3.SOD
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|align="center"|163.1
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|align="center"|1.94
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|}
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====Digestion====
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{|border="1" cellpadding="1" cellspacing="0"
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|&nbsp;
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!width="50"|pSB1C3.<br />SOD
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|-
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|10X FastDigest buffer
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|align="center"|2
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|-
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|DNA (1 &mu;g)
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|align="center"|12.3
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|-
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|dH<sub>2</sub>O
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|align="center"|3.7
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|-
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|FD EcoRI
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|align="center"|1
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|-
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|FD SpeI
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|align="center"|1
 +
|-
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|&nbsp;
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!20 &mu;l
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|}
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*Incubation: 37 &deg;C, 30 min
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 +
====Gel verification====
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[[image:Gelver_dig_pC.SOD_11oct.png|200px|thumb|right|'''Gel verification of pSB1C3.SOD digested with EcoRI and SpeI.'''<br />3 &mu;l &lambda;; 3 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder]]
 +
 +
1 % agarose, 140 V
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'''Expected bands'''
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*Vector: 2175 bp
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*Insert: 495 bp
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'''Results'''<br />
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Band slightly larger than expected, but this may also be due to bound restriction enzymes and/or other disturbances. Proceeded to gel extraction.
 +
 +
====Gel extraction====
 +
Digested pSB1C3.SOD separated on 1 % agarose gel at 110 V, 17 &mu;l sample volume. SOD band extracted and DNA, as well as DNA from samples stored in -20 &deg;C 7/10, were purified using the E.Z.N.A. Gel Extraction kit.
 +
 +
DNA measurements extremely low, but proceeded to ligation/cloning anyway.

Revision as of 12:49, 14 October 2010

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Contents

Andreas

Removal of insertion in BioBrick suffixes

Plasmid prep

From 8/10 stored pellet

  • 50 μl elution buffer.
DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.SOD 163.1 1.94

Digestion

  pSB1C3.
SOD
10X FastDigest buffer 2
DNA (1 μg) 12.3
dH2O 3.7
FD EcoRI 1
FD SpeI 1
  20 μl
  • Incubation: 37 °C, 30 min

Gel verification

Gel verification of pSB1C3.SOD digested with EcoRI and SpeI.
3 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder

1 % agarose, 140 V

Expected bands

  • Vector: 2175 bp
  • Insert: 495 bp

Results
Band slightly larger than expected, but this may also be due to bound restriction enzymes and/or other disturbances. Proceeded to gel extraction.

Gel extraction

Digested pSB1C3.SOD separated on 1 % agarose gel at 110 V, 17 μl sample volume. SOD band extracted and DNA, as well as DNA from samples stored in -20 °C 7/10, were purified using the E.Z.N.A. Gel Extraction kit.

DNA measurements extremely low, but proceeded to ligation/cloning anyway.

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