Team:Stockholm/8 August 2010

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(New page: {{Stockholm/Top2}} ==Andreas==)
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==Andreas==
==Andreas==
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 +
===Cloning===
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 +
====Colony PCR====
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 +
Picked four colonies from 3/8 IgG protease plate (A, B, C & D). Also chose two plasmid samples of pSB1C3.BBa_J18932 (A & B) for PCR verification, to be compared with the amplified yCCS samples from 7/8.
 +
 +
'''PCR tubes'''<br>
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*22.5 &mu;l dH<sub>2</sub>O
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*1 &mu;l VF2
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*1 &mu;l VR
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*0.5 &mu;l cell suspension
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*illustra Ready-to-Go PCR Beads
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 +
::'' '''Negative control (18):''' Blank''
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'''PCR settings'''
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{|border="1" cellpadding="2" cellspacing="0"
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|'''1) Denaturation:'''
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|colspan="2"|95 &deg;C - 10:00
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|-
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|'''2) Denaturation:'''
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|95 &deg;C - 0:30
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|rowspan="3"|x30
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|-
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|'''3) Annealing:'''
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|55 &deg;C - 0:30
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|-
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|'''4) Elongation:'''
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|72 &deg;C - 1:45
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|-
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|'''5) Finish:'''
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|colspan="2"|25 &deg;C - &infin;
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|}
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====Gel verification====
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#1 % agarose, 110 V, 35 min
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#1 % agarose, 90 V, 30 min
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 +
'''Results'''
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#Bands seem identical in size. Highly probable that my yCCS clones are incorrect.
 +
#Strange band sizes, not corresponding at all to what would be expected from IgG protease. I will redo IgG protease cloning next week.
 +
#*Did I forget to load the ladder, or did I load loading dye? That might explain the big blob.

Revision as of 17:54, 8 August 2010

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Contents

Andreas

Cloning

Colony PCR

Picked four colonies from 3/8 IgG protease plate (A, B, C & D). Also chose two plasmid samples of pSB1C3.BBa_J18932 (A & B) for PCR verification, to be compared with the amplified yCCS samples from 7/8.

PCR tubes

  • 22.5 μl dH2O
  • 1 μl VF2
  • 1 μl VR
  • 0.5 μl cell suspension
  • illustra Ready-to-Go PCR Beads
Negative control (18): Blank

PCR settings

1) Denaturation: 95 °C - 10:00
2) Denaturation: 95 °C - 0:30 x30
3) Annealing: 55 °C - 0:30
4) Elongation: 72 °C - 1:45
5) Finish: 25 °C - ∞

Gel verification

  1. 1 % agarose, 110 V, 35 min
  2. 1 % agarose, 90 V, 30 min

Results

  1. Bands seem identical in size. Highly probable that my yCCS clones are incorrect.
  2. Strange band sizes, not corresponding at all to what would be expected from IgG protease. I will redo IgG protease cloning next week.
    • Did I forget to load the ladder, or did I load loading dye? That might explain the big blob.
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