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| - | =PCR and digestion controls= | + | =Microscope investigations of filamentous cells= |
| - | | + | We spent today looking at our filamentous cells under the microscope. |
| - | ==Aims==
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| - | Since we have recently had problems with both PCR and EcoR1 restriction digests, we set up positive controls to ensure that there is nothing wrong with our enzymes or buffers. We performed a single digest of pGFP-rrnb with EcoR1 and PCR of pSB1C3 and a fragment of the ''rocF'' coding sequence from ''Bacillus subtilis'' 168 genomic DNA.
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| - | ===Materials and Protocols===
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| - | Please refer to the [[Team:Newcastle/Restriction_digests|restriction digest]], [[Team:Newcastle/PCR#Phusion_PCR|Phusion PCR]] and [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]] protocols.
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| - | ==...==
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| - | ==Results==
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| - | [[Image:Newcastle_controlgel_270810.jpg|500px|centre]]
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| - | '''Figure 1''': Gel electrophoresis of the amplified PCR products and restriction digest
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| - | *'''Lane 1''': 1 Kb ladder
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| - | *'''Lane 2''': digested (linearised) pGFP-rrnB
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| - | *'''Lane 3''': PCR product of pSB1C3
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| - | *'''Lane 4''': PCR product of first fragment of ''rocF'' coding sequence
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| - | *'''Lane 5''': 1 Kb ladder
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| - | ==Discussion==
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| - | Worked correctly.. nothing is wrong with our enzymes.. in the case of our ''rocF'' plasmid PCRs, it must be the primers which are the problem.. new ones are supposed to be arriving today.. in the other cases, repeat..
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| - | =''yneA''=
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| - | ==Starch plating==
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| - | ===Aims===
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| - | Yesterday we replica plated ''Bacillus subtilis'' 168, that were transformed on Wednesday, onto starch plates.
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| - | Today we aim to see whether or not the transformed ''Bacillus subtilis'' can breakdown starch. If they cannot break down starch there will be no halo when we test with iodine.
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| - | ===Materials===
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| - | *Starch plates
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| - | *Pipette tips
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| - | *Iodine
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| - | ===Results===
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| - | Unfortunately our colonies had halos. Although the insert has integrated, the colonies have antibiotic restistance, the insert has not integrated at the ''amyE'' locus.
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| - | <center>
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| - | {|
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| - | |[[Image:Starchplate.jpg|300px|centre]]
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| - | |[[Image:Starchplate2.jpg|300px|centre]]
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| - | |}
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| - | </center>
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| | {{Team:Newcastle/footer}} | | {{Team:Newcastle/footer}} |
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