IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/18

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Test Ery efficiency
Since 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.


 * First protocol

- 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)


 * Second protocol

- 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)

- Plate cells after 10min of incubation (without DNA) on blank plates (to see if they are alive)

PA with I13522, E0040 and B0034 Preparation
Gel: Result:
 * 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
 * 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
 * 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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