"

From 2010.igem.org

You do not have permission to edit this page, for the following reason:

You can view and copy the source of this page:

__NOTOC__ ===Week6 Highlights=== We continued experiencing significant problems with our 3A assembly. We tried having every teammate attempt a 3A assembly to determine if it was individual error. We also tried using Gold competent cells instead of TOP10 competent cells. All of our assemblies for the week still failed. On a brighter note, we successfully made competent TOP10 cels. ---- ===7/19/10=== None of the assemblies grew. Had 2 different team members retry 3A assembly of CP1-GFP, LacP-GFP, LacPI1-GFP. One team member did not purify digestion products. One team member PCR purified digestion products. Ran a gel of the digestion products --> restriction digest was successful Inoculated TOP10 seed stocks for 2 hours 37C --> OD600nm of 0.25 Made the TOP10 cells competent using the OpenWetWare protocol. ---- ===7/20/10=== None of the assemblies grew. Ran a gel of the ligation products --> ligation was successful, but very little DNA Tried 3A assembly using 50μl of competent TOP10 cells with either 1μl, 5μl, or 10μl of ligation product. Ethanol precipitation of 1-2I, 1-6G, and 2-6K. ---- ===7/21/10=== Transformed and plated TOP10 competent cells using a standard plasmid --> plan to test competency tomorrow Poured 1 sleeve of Amp plates. We came to the conclusion that our old Tet plates either had too little Tet antibiotic or the antibiotic had degraded from exposure to light --> poured 1 sleeve of Tet plates Transformed 5μl of LacPI1-GFP and CP1-GFP ligation product with 25μl of competent TOP10 cells. ---- ===7/22/10=== Weekly meeting with advisers. Went over MAGE knock-out technique. Discussed our problems with 3A assembly. Measured the competence of TOP10 cells --> 9600 cfu/μgDNA Transformed TOP10 competent cells using pUC19 standard plasmid DNA. Plan to measure competence again tomorrow. Streaked EcNR2 cells on Chl and Carbenicillin. Plan to use this strain carry out our MAGE double knock-out. 3A Assembly of CP1-GFP, LacP-GFP, LacPI1-GFP. Ran 2 parallel assemblies - one with a 10 min ligation step and one with a 1 hour ligation step. Transformed using both TOP10 cells and BL21-Gold cells. Kit to stock of 1-2I and 2-6K. Received sponsorship from EMD Chemicals through Millipore. ---- ===7/23/10=== Realized that Chl was never added to our Chl plates --> added 200ul of 1x Chl solution to our old Chl plates to salvage the plates Poured 2 new sleeves of Chl plates. Ran a gel of Chl and Tet linear plasmid backbone PCR product --> successful PCR PCR purified Chl and Tet linear plasmid backbone PCR product. Plated Gold 2ul-ligation product 10min-ligation time (CP1-GFP), Gold 2ul-ligation product 1hr-ligation time (LacP1), Gold 2ul-ligation product 10min-ligation time (LacPI1), Gold 8ul-ligation product 1hr-ligation time (CP1-GFP), Gold 8ul-ligation product 10min-ligation time (LacP1), and Gold 8ul-ligation product 1hr-ligation time (LacPI1) transformed cells. ----

Return to Week6 7/18/10-7/24/10.