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.
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.
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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=First transformation of 'Bacillius subtilis 168' with Prrnb-GFP containing YneA=
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==Aim==
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The aim of the experiment is to perform insert the plasmid Prrnb-GFP 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 antibiotic chloramphenicol and streptomycin, therefore those that have successful intergated will be selected with agar plates that contain these antibiotic. 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 locus. Thus those that have intergardted 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.
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.
"
