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Minnesota/20 June 2008
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| 1. Transformations: Transformations of genes/plasmids into TOP10 E.Coli cells, and onto LB plates showed 1-5 colonies per plate. |
| 2. Primers: Transferring from glass tubes to 1.5mL tubes. All primers were designed in Vector NTI and ordered for synthesis from the BioMedical Genomics center on University of Minnesota's campus. |
| NOTE: Tube #12 looks darker than other primers possibly contaminated. |
| 3. Minprep: Overnight 2mL cultures of colonies picked from 6-18-08 transformations were prepped following Kat's protocols to purify plasmid DNA. |
| a. Cells were spun down @ 10k RPM for 2 minutes in a centrifuge. The supernate was discarded. |
| b. 150uL of minprep solution 1 was added to each tube. After vortexing vigorously, cells were allowed to incubate on ice for 5 minutes. During this time, miniprep solution 2 was prepared. |
| c. Miniprep solution 2 contains: 20uL NaOH (0.2x) + 100uL SDS (10x) + 880uL H20 = 1mL solution. |
| d. 200uL of mp solution 2 was added to the cells, and they were gently mixed by inverting. |
| e. After incubating on ice for 5 minutes, 150uL miniprep solution 3 was added to lyse cells, and tubes were immediately vortexed. AFter another 5 minutes of incubation on ice, cells were spun down for 5 minutes @ 10k RPM in the centrifuge. The supernate was moved to new 2mL tubes, and plasmid was precipitated with 1mL of 100% EtOH (ice cold). |
f. After a 5 minute incubation on ice, DNA was pelleted by centrifuging @ 10k RPM for 10 minutes. The supernate was removed, and the pellet was washed with 70% ethanol. The supernatant was discarded and the pellet was allowed to air dry for 30 minutes in the 37C incubator. After this, pellets were resuspended in TE buffer containing RNaseA (0.5uL RNAse/mL soln), and kept @ 37C for 30 mins. Cells were then stored @ 20C.
 4. Transformations continued: Since colonies had been obtained from parts 1,2,3,6,12,13; transformations of parts 4,5,7,8,9,10,11,14 into TOP10 chemically competent cells was performed (cells provided by Invitrogen). Since there was not enough liquid in the tubes with paper spots from 6-18, 5uL of TE (ph 8.0) was added to each. These were warmed in a 50 degree Celsius H20 bath for 20 minutes while a through c were performed: |
| a. Thaw 50uL aliquots of TOP10 cells cells on ice. |
| b. Add 2uL plasmid/DNA solution to each 50uL aliquot of cells. |
| c. Incubate on ice for 20 minutes. |
| d. Heat shock 1 minute @ 42C, with mixing by flicking tubes every 20 seconds. |
| e. Incubate on ice for 5 minutes. |
| f. Recover in 250uL S.O.C. medium @ 37C with shaking @ 225 rpm's for 2 hours. |
| g. Cells were then plated 100uL/plate and incubate O/N (overnight) @ 37C. |
| *Refer to iGEM “transformation” section for details. |
| NOTE: only #6, #12, and #13 plasmids/genes grew on Dh5alpha competent cells. The other eleven plasmids used had to be re-done for a transformation onto TOP10 competent cells. |
