Team:Stockholm/11 October 2010

From 2010.igem.org

(Difference between revisions)
(→Andreas)
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DNA measurements extremely low, but proceeded to ligation/cloning anyway.
DNA measurements extremely low, but proceeded to ligation/cloning anyway.
 +
 +
====Ligation====
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!pSB1C3.<br />IgGp
 +
!pSB1C3.<br />bGFG
 +
!pSB1C3.<br />ProtA
 +
!pSB1C3.<br />yCCS
 +
!pSB1C3.<br />SOD
 +
|-
 +
|10X T4 Ligase buffer
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|-
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|Vector DNA
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|align="center"|3
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|align="center"|3
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|align="center"|3
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|align="center"|3
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|align="center"|3
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|-
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|Insert DNA
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|align="center"|14
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|align="center"|14
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|align="center"|14
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|align="center"|14
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|align="center"|14
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|-
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|dH<sub>2</sub>O
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|align="center"|0
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|align="center"|0
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|align="center"|0
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|align="center"|0
 +
|align="center"|0
 +
|-
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|T4 DNA ligase
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|align="center"|1
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|align="center"|1
 +
|align="center"|1
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|align="center"|1
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|align="center"|1
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|-
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|&nbsp;
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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|}
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*Incubation: 22 &deg;C, 15 min
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 +
====Transformation====
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*Mod. quick transformation
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**Top10
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**3 &mu;l ligation mix
 +
**30 min incubation on ice

Revision as of 13:41, 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.

Ligation

  pSB1C3.
IgGp 		pSB1C3.
bGFG 		pSB1C3.
ProtA 		pSB1C3.
yCCS 		pSB1C3.
SOD
10X T4 Ligase buffer 2 2 2 2 2
Vector DNA 3 3 3 3 3
Insert DNA 14 14 14 14 14
dH2O 0 0 0 0 0
T4 DNA ligase 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

  • Mod. quick transformation
    • Top10
    • 3 μl ligation mix
    • 30 min incubation on ice
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