Team:Toronto/Notebook-w07-mon
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Monday June 27, 2016
Monday, 6/27
Members Present: Hamed, Alex, Cathy, Celine, Kat, Bohdan, Andrea, Davesh
LAB:
Morning:
- Restriction enzyme digested the backbone and Short Linear Tet-O - LacZ alpha (from the parts that were PCR amplified on friday)
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PCR Purified the digested parts, then nanodropped:
A | B | |
1 | Sample | Concentration |
2 | BB (backbone) | 4.8 ng/ul |
3 | Lac-Z -1 | 9.4 ng/ul |
4 | LacZ -2 | 1.8 ng/ul |
Table1
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Proceeded to ligation
Afternoon:
- Ran the following gel:
GEL: 1. PCR amplified pSB1C3 backbone. 2. PCR amplified Short Linear Tet-O LacZ-alpha. 3. Restriction enzyme (EcoRI, PstI, DpnI) digested bacbone. 4. Restriction enzyme (EcoRI, PstI) digested Short Linear Tet-O LacZ-alpha, first attempt. 5. Restriction enzyme (EcoRI, PstI) digested Short Linear Tet-O LacZ-alpha, second attempt. 6. Further PCR purified pSB1C3 backbone. 7. 3kb DNA ladder. 8. Further PCR purified short Linear Tet-O LacZ-alpha, attempt 1. 9. Further PCR purified short Linear Tet-O LacZ-alpha, attempt 2. 10. Ligated construct, attempt 1. 11. Ligated construct, attempt 2. 12. Miniprepped RFP.
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Considering that the gel did not work beyond the PCR phase, we suspect that something is amiss with the PCR process. Potentially the Phusion polymerase, which was left over from last year.
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Transformation with the two ligation samples, RFP, E.glowli (bioluminescence reporter from the iGEM distributionkill plate 1, well 4L), as well as a no plasmid control.
Administrative:
TO DO:
For the next day:
LAB TEAM:
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Trouble shoot the PCR reaction for thebackbone and for the Lac-Z alpha fragment. (this means trying fresh reagents, potentially running gradients)
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Resuspend all of our construct and consider doing a bulk PCR, with some gradients pending on the availablity of reagents.
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Making a new batch of RbCl competent cells in the cold room.
LAB MANAGERS: Buy 2 respirators
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Stock up on PCR reagents that we may be running low on:
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Especially - NEB Phusion polymerase.
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Might need another DNA ladder for running gels in the future. Consult with other members for appropriate size
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I NEED USB PLEASE
Gmail correspondence:
Meetings/Notes:
References:
