Team:Newcastle/27 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=PCR and digestion controls=
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=Microscope investigations of filamentous cells=
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We spent today looking at our filamentous cells under the microscope.
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==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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==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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The single digest and two PCR reactions both worked correctly.
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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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|-
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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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=''yneA''=
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==PCR (Repeat)==
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===Aim===
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To repeat the PCR that we did [[Team:Newcastle/25_August_2010|yesterday]] using the correct ''rocF'' primers.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/PCR|PCR]].
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==PCR Purification==
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===Aim===
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To remove unwanted primers, taq polymerase, buffer and salts to obtain pure DNA.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/PCR_purification|PCR purification]].
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==Digestion==
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===Aim===
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To digest the PCR products of pSB1C3 and ''yneA'' from PCR purification.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
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==Gel extraction==
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===Aim===
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To purify the DNA of ''yneA'' and pSB1C3 by extracting the bands from the gel after running gel electrophoresis. Concentration of DNA is then checked with NanoDrop.
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===Materials and Protocol===
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Please refer to:
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* [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]],
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* [[Team:Newcastle/Gel_extraction|gel extraction]] and
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* [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]].
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===Results===
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When we look at the gel through the GelDoc, only pSB1C3 was successfully digested with EcoR1 and Pst1 while ''yneA'' did not show any bands.
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===Conclusion===
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We will repeat the protocol for ''yneA'' on [[Team:Newcastle/31_August_2010|31.08.10]].
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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 01:17, 28 October 2010

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Microscope investigations of filamentous cells

We spent today looking at our filamentous cells under the microscope.

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