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| - | =pH changing= | + | =Preparation for pH acclimatisation of ''Bacillus subtilis'' 168= |
| - | We brought hepes (which is similar to homopipes) to pH 7 then autoclaved it.
| + | We prepared 100mM of HEPES (it is a dibasic compound) at pH 7.0 and sent it for autoclaving. |
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| - | =Preparing medium for natto=
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| - | We prepared antibitoic ,medium for natto: 17.5g of broth in 1L, so 1.75 in 100ml
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| - | =Extreme Overnight cultures for Gibson=
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| - | # G1 (T1) 1 Colony 1,
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| - | # G1 (T2) 7 Colonies 2-6
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| - | # G2 (T3) 2 Colonies 9,10
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| - | # G2 (T4) 14 Colonies 11-24
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| - | # G3 (T5) 3 Colonies 25-27
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| - | # G3 (T6) 2 Colonies 28-29
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| - | # G4 (T7) 4 Colonies 30-33
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| - | # G4 (T8) 9 Colonies 34-42
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| - | # G5 (T9) 0
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| - | # G5 (T10) 2 Colonies 43-44
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| - | =Gibson for ''rocF''=
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| - | *Promoter 136bp - 0.7 microl
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| - | * rocF 1 246bp - 1.2 microl
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| - | * rocF 2 597bp - 1.9 microl
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| - | * rocF 3 125bp - 0.4 microl
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| - | * DTERM 116bp - 0.8microl
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| - | Total: 1190bp
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| - | =Transformation of ''Bacillius subtilis'' 168 with pGFP-rrnB containing ''yneA''=
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| - | ==Aim==
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| - | The aim of the experiment is to perform insert the plasmid pGFP-rrnB containing ''yneA'' which have been ligated eariler into the chromosome of ''Bacillus subtilis'' 168. ''B. subtilis'' containing the intergated vector will be resistance to both the antibiotics chloramphenicol and streptomycin, therefore those that have successful intergated will be selected with agar plates that contain both the antibiotics. The second step will be to identify those colones that have the plasmid intergated at the corerct position in the chromosome, which is the amylase gene locus. Thus those that have intergrated at the wrong position will not be able to break down starch, which can be tested with the iodine test.
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| - | ==Materials and Protocol==
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| - | Please refer to: [[Team:Newcastle/Transformation of B. subtilis| Transformation of ''Bacillus subtilis'']]
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| - | Note: Overnigth culture of ''B. subtilis 168'' in MM competence medium was done the day before and the iodine test was performed the day after transformation.
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| | {{Team:Newcastle/footer}} | | {{Team:Newcastle/footer}} |
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