IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/18
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- Plate cells after 10min of incubation (without DNA) on blank plates (to see if they are alive) | - Plate cells after 10min of incubation (without DNA) on blank plates (to see if they are alive) | ||
| + | == PA with I13522, E0040 and B0034 Preparation== | ||
| + | |||
| + | * R0040 TOP10 miniprep from LB culture done by Kevin | ||
| + | * 5 single colonies of I13522 picked onto Amp100 plate and LB with Amp100 done on Sunday | ||
| + | * Miniprep of the 5 colonies with sinigle colony PCR | ||
| + | * PCR followed standard protocol and product is run on 0.8% EtBr E-gel | ||
| + | * iGEM34 porgramme is used for PCR | ||
| + | Gel: | ||
| + | ** 4μL dye with 20μL PCR product | ||
| + | ** 5μL Hyperladder I with 15μL SDW into lane 2 | ||
| + | ** Lanes 3-7: I13522 Colonies 1-5 | ||
| + | ** Lane 8: R0040 | ||
| + | ** Lane 9: Hyperladder I | ||
| + | Result: | ||
| + | * Size of I13522 is not right as the bands are all around 4500bp but it should be 2375bp | ||
| + | * R0040 is of the correct size | ||
| + | |||
| + | * Transformed chemically compotent TOP10 with B0034 biobrick extract and pUC9 control | ||
| + | * Plate on Amp100 plates after standard transformation | ||
| + | |||
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Revision as of 06:56, 28 October 2008
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Test Ery efficiencySince all Ery plates grew, we want to check the antibiotic stock. - New Ery plate of IA751 (they should die) - New LB stock (Ery5) with IA751 Single colony plates- New plates (Cm5 + Amp100) with single colony of ECE188 (from 14/08), ECE189 (from 13/08) and ECE190 (from 14/08) - Grow the same single colony into LB with antibiotic Transformation of glycerol stocks of B.S.The result from the last transformation of glycerol stocks was negative> We tried again with two different protocols.
- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B - Incubate about 1h15 - Add 0.6μg of ECE166 (because we managed to transform BS with this vector) - Incubate 1h30 - Plate on Cm5 plates (DNA less control and transformed cells)
- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B - Add 0.6μg of ECE166 (because we managed to transform BS with this vector) - Incubate 30min - Plate on Cm5 plates (transformed cells)
PA with I13522, E0040 and B0034 Preparation
Gel:
Result:
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