Team:Newcastle/26 August 2010

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{{Team:Newcastle/mainbanner}}
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=''yneA''=
 
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==PCR (Repeat)==
 
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===Aim===
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=Screening ''Bacillius subtilis'' transformants=
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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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==Aim==
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The aim of the experiment is to identify those colones that have the plasmid integrated at the correct position in the chromosome, which is the ''amyE'' locus. Those that have integrated at the correct position will not be able to break down starch, which can be tested by exposing the colonies on the starch plates to iodine.
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===Materials and Protocol===
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===Results===  
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Please refer to [[Team:Newcastle/PCR|PCR]].
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Some of our colonies did not have halos, therefore the transformation and integration was successful for these colonies.
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Below you can see cells from these positive colonies under the microscope.
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==PCR Purification==
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{|
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|-
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|[[Image:Starchplate.jpg|thumb|250px|centre]]
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| 
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|[[Image:Starchplate2.jpg|thumb|250px|centre]]
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|}
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===Aim===
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{|
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|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|250px|centre]]
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To remove unwanted primers, taq polymerase, buffer and salts to obtain pure DNA.
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|
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|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |250px|centre]]
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===Materials and Protocol===
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|
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|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|250px|centre]]
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Please refer to [[Team:Newcastle/PCR_purification|PCR purification]].
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|}
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==Restriction Digest==
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===Aim===
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To digest the PCR products of pSB1C3 and ''yneA'' from PCR purification with enzymes EcoR1 and Nhe1.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
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===Results, Discussion and Conclusion===
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We run the digested products with gel electrophoresis to determine whether the digest worked.
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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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The gel did not show any band when we looked at it from GelDoc.
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===Conclusion===
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The restriction digest did not work, so we will repeat the protocol again [[Team:Newcastle/27_August_2010|tomorrow]].
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Latest revision as of 01:15, 28 October 2010

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Screening Bacillius subtilis transformants

Aim

The aim of the experiment is to identify those colones that have the plasmid integrated at the correct position in the chromosome, which is the amyE locus. Those that have integrated at the correct position will not be able to break down starch, which can be tested by exposing the colonies on the starch plates to iodine.

Results

Some of our colonies did not have halos, therefore the transformation and integration was successful for these colonies.

Below you can see cells from these positive colonies under the microscope.

Starchplate.jpg
Starchplate2.jpg
Filamentous cells
Filamentous cells showing GFP signal
Normal Bacillus subtilis 168



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