Team:Newcastle/27 July 2010

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===Aims of genomic DNA extraction experiment===
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=Genomic DNA extraction experiment=
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The aim of today's experiment is to extract genomic DNA from ''Bacillus subtilis'' strain 3610,genes from which will be needed for the swarming biobrick.
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====Materials Required====
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[[Image:Newcastle alan chromosome.jpg|thumb|200px|right]]
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* Cells grown from yesterday
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[[Image:Newcastle ice chromosome.jpg|thumb|200px|right]]
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* Centrifuge
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==Aims==
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* pipette
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The aim of today's experiment is to extract genomic DNA from both ''B. subtilis'' strains 168 and 3610. The genes necessary for the [[Team:Newcastle/Swarming|swarming BioBrick]] and [[Team:Newcastle/Urease|''rocF'' BioBrick]] will then hopefully be obtained from the genomic DNA using PCR.
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* lysozyme
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* Cell lysis solution
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* RNase solution
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* protein precipitation solution
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* ice
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* isopropanol
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* ethanol
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====Procedure====
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==Protocol==
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* Please refer to: [[Team:Newcastle/DNA extraction| DNA extraction of ''B. subtilis'']] for materials required and protocol.
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=====Cell lysis=====
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==Discussion==
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# Pellet cells by centrifugation at 3600rpm for 10 minutes.
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At the end of the DNA precipitation step, we observed a small white pellet in all the eppendorf tubes.
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# Pour off supernatant.
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# Add 0.5ml of cell suspension solution, gently pipet up and down to resuspend and transfer to 1.5 ml eppendorf tube.
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# Add 25microlitres of lysozyme and invert 25 times.
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# Incubate for 30 minutes at 37°C inverting occasionally.
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# Centrifuge at 13000rpm for 10minutes to pellet the cells then remove the supernatant.
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# Add 0.5ml of cell lysis solution to the cell pellet and gently pipet up and down to lyse the cells.
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# Heat sample for 30 minutes mix every 5-10 minutes.
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=====RNase treatment=====
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==Conclusion==
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# Add 3 microlitres of RNase A solution to the cell lysate
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The experiment was a success! The quality of the extracted DNA will be checked by using PCR on 28th July, 2010.
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# Mix by inverting 25 times and incubate at 37°C for 60 minutes
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=====Protein precipitation=====
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=Preparation for cloning of the rocF BioBrick=
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# Cool samples on ice.
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==Results==
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# Add 0.5 ml protein precipitation solution to each tube.
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# Vortex vigorously at high speed for 20 seconds to miux the protein precipitation solution uniformly with the cell lysate. Place samples on ice for 5 minutes.
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# Centrifuge at 13000 rpm for 30 seconds or until the precipitated proteins form a tight pellet.
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=====DNA precipitation=====
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# Pour the supernatant containing the DNA into a clean eppendorf tube. (The samples may be kept at -20°C overnight at this stage.)
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# Add 0.5ml isopropanol to each tube.
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# Mix by inverting gently for 50 times.
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# Centrifuge at 13000 rpm for 1 minute. The DNA should be visible as a small white pellet.
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# Pour off the supernatant and drain the tube on clean absorbent paper. Add 0.5 ml 70% ethanol and invert tube several times to wash the DNA.
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# Centrifuge at 13000 rpm for 1 minute. Carefully pour off the ethanol.
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# Drain the tubes on clean absorbent paper. Allow to air dry for 10-15 minutes.
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=====DNA hydration=====
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# Add 100 µl DNA hydration solution to each tube.
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# Rehydrate DNA by incubating the sample for 1 hour at 65°C and overnight at room temperature. Tap the tube periodically to aid in dispersing the DNA.
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# For storage, centrifuge briefly and store at -20°C.
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====Discussion====
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At the end of the DNA rpecipitation step, we did not observe any small white pellet.
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====Conclusion====
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If the experiment has failed the experiment will be redone on Monday, 26th July, 2010.
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Yesterday, we transformed ''E. coli'' DH5α with pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]. After checking the plates today for colonies we observed lots of pink colonies on the pSB1C3 plates and numerous white colonies on the [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plates. Plates were then stored at 4°C as colonies will be used for minipreps.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 22:12, 27 October 2010

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Contents

Genomic DNA extraction experiment

Newcastle alan chromosome.jpg
Newcastle ice chromosome.jpg

Aims

The aim of today's experiment is to extract genomic DNA from both B. subtilis strains 168 and 3610. The genes necessary for the swarming BioBrick and rocF BioBrick will then hopefully be obtained from the genomic DNA using PCR.

Protocol

Discussion

At the end of the DNA precipitation step, we observed a small white pellet in all the eppendorf tubes.

Conclusion

The experiment was a success! The quality of the extracted DNA will be checked by using PCR on 28th July, 2010.

Preparation for cloning of the rocF BioBrick

Results

Yesterday, we transformed E. coli DH5α with pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]. After checking the plates today for colonies we observed lots of pink colonies on the pSB1C3 plates and numerous white colonies on the [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plates. Plates were then stored at 4°C as colonies will be used for minipreps.

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