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

- 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

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)

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

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:

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