August 30th, 2010
- Transformation with L2: p30-MinBP + GFP BBa_K145015 (74 min) made on August 26 was not succesful because cells didn’t grow as expected, it may be because we’ve been using pSB4A5, which is a low copy plasmid (~5 copies per cell). I’ll re-do the transformation again but using a lower concentration of Ampicilin.
- After transformation I’ll perform colony PCR to test the ligation is correct and if so, plasmid extraction.
- After plasmid extraction and ligation test, I’ll amplify the BluePromoter+RBS+GFP from the extracted plasmid to ligate this construction into pSB3K3.
- Once inserted into pSB3K3 I’ll transform this new construction on DH5-α and TOP10, also I’ll transform on these two strains BBa_I20260 and BBa_I20260 with the changed RBS.
1. Transform L2: p30-MinBP + GFP BBa_K145015 (74 min) and p30 (as positive control) into DH5-α competent cells.
- Unfreeze competent cells (keep on ice).
- Add 5ul of exogenous DNA to 100ul of compentent cells.
- Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
- Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
- Incubate 5 min on ice.
- Transfer the cells to a tube with 1ml of LB medium
- Incubate the cells in the LB medium for 1hr at 37ºC and shaking (during this period the cells recover their metabolic activity, replicate and synthesize the proteins encoded in the material just inserted).
- Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
- Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
- Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
- Incubate the plates at 37ºC overnight.
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