Team:Newcastle/26 August 2010

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(New page: =''yneA''= ==PCR (Repeat)== ===Aim=== To repeat the PCR that we did yesterday using the correct ''rocF'' primers. ===Materials and Protocol=== Please...)
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=''yneA''=
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{{Team:Newcastle/mainbanner}}
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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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=Screening ''Bacillius subtilis'' transformants=
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===Materials and Protocol===
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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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Please refer to [[Team:Newcastle/PCR|PCR]].
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===Results===
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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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==PCR Purification==
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Below you can see cells from these positive colonies under the microscope.
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===Aim===
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{|
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|-
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|[[Image:Starchplate.jpg|thumb|250px|centre]]
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|[[Image:Starchplate2.jpg|thumb|250px|centre]]
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|}
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{|
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|[[Image:Newcastle_filamentous_pc_expt1.jpg|thumb|Filamentous cells|250px|centre]]
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|[[Image:Newcastle_filamentous_gfp_expt1.jpg|thumb|Filamentous cells showing GFP signal |250px|centre]]
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|[[Image:Newcastle_filamentous_control_pc_expt1.jpg|thumb|Normal ''Bacillus subtilis ''168|250px|centre]]
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|}
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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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{{Team:Newcastle/footer}}
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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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The bands we got from gel electrophoresis is very faint.
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===Conclusion===
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We realized that we used the wrong ''rocF'' primer, so we repeat the whole procedure from PCR again.
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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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