Virginia Tech/sequencing
From 2007.igem.org
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To aid in quality control, our lab undertook a sequencing project of the entire registry. The following procedure was conducted: | To aid in quality control, our lab undertook a sequencing project of the entire registry. The following procedure was conducted: | ||
| - | The | + | The iGEM construct plasmids were resuspended in 30 of nuclease free water (Ambion) at 4°C overnight. They were quantified using the Nanodrop spectrophotometer. 20ng of Plasmid DNA was used in the PCR amplification of the iGEM construct, using Qiagen’s Taq PCR master mix kit, and 2uM BioBrick forward and reverse primers at 100ul reaction volume. |
The PCR product was purified using Qiagen’s QIAquick PCR purification kit and resuspended in 25ul of nuclease free water, and QC’d on the Agilent Bioanalyser DNA 7500 assay. The Amplified product was the quantified and diluted to 10ng per ul. The PCR product along with the biobrick primers were submitted to the VBI Core Laboratory for Sanger sequencing. | The PCR product was purified using Qiagen’s QIAquick PCR purification kit and resuspended in 25ul of nuclease free water, and QC’d on the Agilent Bioanalyser DNA 7500 assay. The Amplified product was the quantified and diluted to 10ng per ul. The PCR product along with the biobrick primers were submitted to the VBI Core Laboratory for Sanger sequencing. | ||
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PCR conditions | PCR conditions | ||
| - | + | 94°C 45 sec | |
| - | + | 94°C 30 sec | |
| - | + | 55°C 45 sec | |
| - | + | 72°C 45 sec | |
add 24 cycles | add 24 cycles | ||
| - | + | 72C°5 minutes | |
| - | + | 4°C hold | |
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| - | The sequencing data will be made available | + | The sequencing data will be made available [http://www.genocad.org/briobricks.tar.gz here]. |
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Revision as of 22:12, 25 October 2007
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To aid in quality control, our lab undertook a sequencing project of the entire registry. The following procedure was conducted: The iGEM construct plasmids were resuspended in 30 of nuclease free water (Ambion) at 4°C overnight. They were quantified using the Nanodrop spectrophotometer. 20ng of Plasmid DNA was used in the PCR amplification of the iGEM construct, using Qiagen’s Taq PCR master mix kit, and 2uM BioBrick forward and reverse primers at 100ul reaction volume. The PCR product was purified using Qiagen’s QIAquick PCR purification kit and resuspended in 25ul of nuclease free water, and QC’d on the Agilent Bioanalyser DNA 7500 assay. The Amplified product was the quantified and diluted to 10ng per ul. The PCR product along with the biobrick primers were submitted to the VBI Core Laboratory for Sanger sequencing. PCR conditions 94°C 45 sec 94°C 30 sec 55°C 45 sec 72°C 45 sec add 24 cycles 72C°5 minutes 4°C hold
Sequencing conditions 400ng template DNA 3.2 pmol primer 2.5 ul BigDye Terminator mix v3.1 Water to a total volume 15ul
The sequencing data will be made available [http://www.genocad.org/briobricks.tar.gz here].
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