IGEM:Cambridge/2008/Protocols: Difference between revisions
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<div id="about"> | <div id="about"> | ||
{{Cambridge08}} | {{Cambridge08}} | ||
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=Agarose Gel= | =Agarose Gel= | ||
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[[Image:DSC00022.JPG|right|thumb|190px|Agarose gel]] | [[Image:DSC00022.JPG|right|thumb|190px|Agarose gel]] | ||
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Used for Electrophoresis when extracting any product afterwards. | Used for Electrophoresis when extracting any product afterwards. | ||
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=E-Gel= | =E-Gel= | ||
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[[Image:DSC00020.JPG|right|thumb|150px|Our E-gel machine from invitrogen]] | [[Image:DSC00020.JPG|right|thumb|150px|Our E-gel machine from invitrogen]] | ||
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E-Gel is used to confirm size of prodicts, but not for extraction. | E-Gel is used to confirm size of prodicts, but not for extraction. | ||
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=Flame Photometry= | =Flame Photometry= | ||
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[[Image:Chris and Flame Photometer.jpg|20px|frame|right|Chris attempts to unravel the secrets of the Flame Photometer]] | [[Image:Chris and Flame Photometer.jpg|20px|frame|right|Chris attempts to unravel the secrets of the Flame Photometer]] | ||
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'''Set Up''' | '''Set Up''' | ||
:*Make sure discharge pipe (on right hand side) is placed in something to collect discharge, and valve is not completely shut. | :*Make sure discharge pipe (on right hand side) is placed in something to collect discharge, and valve is not completely shut. | ||
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=Plasmid Miniprep (Zyppy)= | =Plasmid Miniprep (Zyppy)= | ||
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[[Image:DSC00021.JPG|right|thumb|240px|Plasmid Miniprep kit]] | [[Image:DSC00021.JPG|right|thumb|240px|Plasmid Miniprep kit]] | ||
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:*Add 100μL 7x Lysis Buffer to 600μL Cell Culture. Invert 4 to 6 times | :*Add 100μL 7x Lysis Buffer to 600μL Cell Culture. Invert 4 to 6 times | ||
:*Add 350μL cold Neutralization Buffer. Invert 4 to 6 times | :*Add 350μL cold Neutralization Buffer. Invert 4 to 6 times | ||
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=PCR= | =PCR= | ||
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[[Image:Piko_pcr.jpg|thumb|220px|right|Our Piko PCR machine provided by Finnzymes]] | [[Image:Piko_pcr.jpg|thumb|220px|right|Our Piko PCR machine provided by Finnzymes]] | ||
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To make 50μL of PCR mix the following were added to Eppendorf tubes: | To make 50μL of PCR mix the following were added to Eppendorf tubes: | ||
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=Transformation= | =Transformation= | ||
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[[Image:DSC00023.JPG|230px|right|thumb|Plates with transformed cells]] | [[Image:DSC00023.JPG|230px|right|thumb|Plates with transformed cells]] | ||
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First chemically competent cells are made | First chemically competent cells are made | ||
:*Cells are grown for 1 hour in LB and pelleted (at 6500rpm for 1 min.) | :*Cells are grown for 1 hour in LB and pelleted (at 6500rpm for 1 min.) |
Revision as of 14:55, 19 August 2008
Agarose Gel
Used for Electrophoresis when extracting any product afterwards. Making the Gel
Loading the Gel
Running the Gel
Visualising the Gel
Biobrick ExtractionWe found we could not extract enough DNA from the registry, so we are upgrading the extraction to the "bigger, better, faster, stronger" method.
We then confirmed with PCR. Competent Cells StocksChemically competent cells are made by growing overnight in 200ml LB in a shaking incubator at 37°C. 200ml are spun down at 3800rpm for 8 mins, resuspended in 20ml CaCl2, spun down again and resuspended in 4ml CaCl2. Again spun down (6500 rpm for 5 mins)and resuspended in 4ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they are aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. To recover cells thaw the tubes on ice, spin down and resucpend in 100μL ice cold CaCl2. Electroporation competent TOP10 and DH5α cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C. 100ml were spun down at 3800rpm for 8 mins, resuspended in 25ml SDW, spun down again and resuspended in 2ml SDW. Again they were spun down (6500 rpm for 5 mins)and resuspended in 2ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. To recover cells thaw the tubes on ice, spin down and resucpend in 100μL ice cold SDW. E-Gel
E-Gel is used to confirm size of prodicts, but not for extraction.
Fast DigestionThe following are added to a 1.5μL eppendorf:
If using PCR product the whole is made up to 30μL, using 10μL DNA and 3μL buffer. The tubes are then incubated at 37°C for 5mins and then the enzyme inactivated at 80°C for 15mins Fast LigationThe following are added to a 1.5μL eppendorf:
The tubes are then incubated at room temperature for 5mins and then the enzyme inactivated at 75°C for 15mins We have now got a different fast ligation kit. Now the following are added to a 1.5μL eppendorf:
The tubes are then incubated at room temperature for 5mins. Flame Photometry
Set Up
Taking readings
In case of compressor blow-out
In case of wildly fluctuating "zero" readings
Gel DNA Recovery (Zymoclean)
Oxygen ElectrodeFor the protocol see http://www.rankbrothers.co.uk/download/digioxy.pdf Plasmid Miniprep (Zyppy)
Creating a Glycerol Stock (Modified Version)
Primer Preparation and StorageSigma Primers arrived as tubes of dried oligos
PCR
To make 50μL of PCR mix the following were added to Eppendorf tubes:
These values can be adjusted for any volumes of final mixture. The total final volume is transferred to thin walled tubes (may need to be spun down) and then placed in a PCR machine and the appropriate program run. Transformation
First chemically competent cells are made
To transform cells
After transformation cells need to be grown under selection conditions. |