Team:Stockholm/12 July 2010

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== Andreas ==
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=== Clonings ===
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==== Gel extractions and DNA clean-up ====
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''Continued from [https://2010.igem.org/Team:Stockholm/12_July_2010 9/7]''
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 +
Gel extracted DNA samples (pEX, BBa_J1893x, SOD and yCCS) and digested DNA samples (pSB1x3) stored in -20°C were purified using the E.Z.N.A. Gel Extraction kit.
 +
 +
Nanodrop measurements revealed extremely low or even nonexistent concentrations of DNA in purified samples. I decided to go ahead with the ligations anyway, as I was confident that there was indeed (correct) DNA present in the samples.
 +
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==== Ligations ====
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:'''Ligation strategy:'''
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:{|border="1" cellspacing="0" cellpadding="2"
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|'''Vector'''
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|'''Insert'''
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|-
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|pSB1A3
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|BBa_J18930
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|-
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|pSB1A3
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|BBa_J18931
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|-
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|pSB1A3
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|BBa_J18932
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|-
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|pSB1A3
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|SOD
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|-
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|pSB1C3
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|BBa_J18930
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|-
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|pSB1C3
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|BBa_J18931
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|-
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|pSB1C3
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|BBa_J18932
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|-
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|pSB1C3
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|SOD
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|-
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|pSB1C3
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|yCCS
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|-
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|pSB1K3
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|BBa_J18930
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|-
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|pSB1K3
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|BBa_J18931
 +
|-
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|pSB1K3
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|BBa_J18932
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|-
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|pSB1K3
 +
|SOD
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|-
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|pEX
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|BBa_J18930
 +
|-
 +
|pEX
 +
|BBa_J18931
 +
|-
 +
|pEX
 +
|BBa_J18932
 +
|-
 +
|pEX
 +
|SOD
 +
|-
 +
|pEX
 +
|yCCS
 +
|}
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:'''Procedures'''
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::''All insert samples, as well as the pEX vector sample, were concentrated in a vacuum evaporator prior to ligation''
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::* 2 μl vector
 +
::* 5 μl insert
 +
::* 10 μl 2X Quick Ligase buffer
 +
::* 2 μl dH2O
 +
::* 1 μl Quick Ligase
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::Incubation 10 min in "RT" (30°C due to the extreme summer heat these days!)
 +
 +
==== Transformations ====
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Ligated constructs transformed into Top10 and plated onto LB agar plates with relevant antibiotics. pEX.BBa_J1893x constructs also transformed into BL21 cells and plated onto 100 μg/ml Amp with 1 % glucose. Plates incubated ON in 37°C.

Revision as of 23:10, 14 July 2010

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Contents


Andreas

Clonings

Gel extractions and DNA clean-up

Continued from 9/7

Gel extracted DNA samples (pEX, BBa_J1893x, SOD and yCCS) and digested DNA samples (pSB1x3) stored in -20°C were purified using the E.Z.N.A. Gel Extraction kit.

Nanodrop measurements revealed extremely low or even nonexistent concentrations of DNA in purified samples. I decided to go ahead with the ligations anyway, as I was confident that there was indeed (correct) DNA present in the samples.

Ligations

Ligation strategy:
Vector Insert
pSB1A3 BBa_J18930
pSB1A3 BBa_J18931
pSB1A3 BBa_J18932
pSB1A3 SOD
pSB1C3 BBa_J18930
pSB1C3 BBa_J18931
pSB1C3 BBa_J18932
pSB1C3 SOD
pSB1C3 yCCS
pSB1K3 BBa_J18930
pSB1K3 BBa_J18931
pSB1K3 BBa_J18932
pSB1K3 SOD
pEX BBa_J18930
pEX BBa_J18931
pEX BBa_J18932
pEX SOD
pEX yCCS
Procedures
All insert samples, as well as the pEX vector sample, were concentrated in a vacuum evaporator prior to ligation
  • 2 μl vector
  • 5 μl insert
  • 10 μl 2X Quick Ligase buffer
  • 2 μl dH2O
  • 1 μl Quick Ligase
Incubation 10 min in "RT" (30°C due to the extreme summer heat these days!)

Transformations

Ligated constructs transformed into Top10 and plated onto LB agar plates with relevant antibiotics. pEX.BBa_J1893x constructs also transformed into BL21 cells and plated onto 100 μg/ml Amp with 1 % glucose. Plates incubated ON in 37°C.

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