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Minnesota/20 June 2008
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4. '''Transformations continued:''' | 4. '''Transformations continued:''' | ||
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|a. Thaw cells on ice | |a. Thaw cells on ice | ||
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Revision as of 16:34, 1 July 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. |
| 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.
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| a. Thaw cells on ice |
| b. Combine & incubate on ice (20 mins); 50uL of cells and 2uL plasmid. |
| c. Heat shock 1 minute @ 42C. |
| d. Incubate on ice for 5 minutes. |
| e. Recover in 250uL S.O.C. @ 37C for 2 hours |
| f. Plate 100uL 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. |
