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Team:British Columbia/Week9
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====beta-pinene & (-)-limonene synthase==== | ====beta-pinene & (-)-limonene synthase==== | ||
*Verified yesterday's SDM-PCR products through gel electrophoresis. No bands were present in any of the lanes. | *Verified yesterday's SDM-PCR products through gel electrophoresis. No bands were present in any of the lanes. | ||
| - | *Troubleshooting: The dNTP could have been too old; the concentrations for the reagents used were not optimized - Marianne researched protocols and optimized the SDM-PCR protocol. We (Vicki and Marianne) will test it tomorrow. | + | *Troubleshooting: The dNTP could have been too old/contaminated (we had troubles with this particular tube of dNTP from last year); the concentrations for the reagents used were not optimized - Marianne researched protocols and optimized the SDM-PCR protocol. We (Vicki and Marianne) will test it tomorrow. |
=Thursday= | =Thursday= | ||
Latest revision as of 16:46, 30 June 2011
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Week 9: Monday June 27 - Sunday July 3
Contents |
Monday
We created a set of lab rules due to common mistakes made in the lab over the past few weeks.
Vicki and Marianne will having training sessions throughout the week to show team members without much wetlab experience how to PCR, cast and run a gel, digest, ligate, transform, design primers, send miniprepped products to be sequenced.
Competent cells - tested, and they're competent! Good job Gurpal and Sam!
Modeling: Jacob obtained the ARC program. Gurpal wants to create a wetlab flow chart to match the modeling.
Sent DspB (part from last year) to Grinnell College (a new iGEM team this year who requested our part) in the United States.
alpha-pinene synthase
- SDM-PCR worked! (Used the QuikChange SDM Kit protocol).
3-carene synthase
Daisy needs to make custom primers for sequencing purposes because the mutagen site is in the middle of the coding sequence and her flanking primers will only sequence 700-800bp into the sequence.
beta-pinene synthase
- Resuspended SDM forward and reverse primers
(-)-limonene synthase
- Resuspended SDM forward and reverse primers
Tuesday
Training: went over PCR protocol (SDM specific)
beta-pinene & (-)-limonene synthase
- Ran SDM-PCR
3-Carene
Daisy did a single digest with EcoRI-HF to test if the EcoRI site is in the synthase or not. Chris Keeling emailed Daisy the sequence he used for the truncation of the 3-Carene synthase. Daisy ordered primers to do the sequencing. However, Alina has not replied to her email to confirm that the primers are ordered.
Wednesday
Training: The plan was to make kanamycin plates, but this was at a stand-still as we had troubles locating the tube of kanamycin sulfate salt.
beta-pinene & (-)-limonene synthase
- Verified yesterday's SDM-PCR products through gel electrophoresis. No bands were present in any of the lanes.
- Troubleshooting: The dNTP could have been too old/contaminated (we had troubles with this particular tube of dNTP from last year); the concentrations for the reagents used were not optimized - Marianne researched protocols and optimized the SDM-PCR protocol. We (Vicki and Marianne) will test it tomorrow.
Thursday
Training:
