IGEM:Groningen/Notebook/iGEM 2011/2011/05/18: Difference between revisions

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==18-5-11==
==18-5-11==
<br>  
<br>  
<br> <br> Protocols for transforming biobricks in cells on http://partsregistry.org/Help:Spring_2011_DNA_distribution
<br> 2PCRs failed yesterday, byproducts
<br>
<br> Try to resuspend them better and leave them on ice after pipetting
<br>DNA Kit Plate Instructions
<br>To use the DNA in the Distribution Kit you may follow these instructions:
<br>
<br>
<br>1.With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you <br>want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to <br>cross contamination between the wells.
<br>2.Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure <br>the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
<br>3.Transform 1 or 2uL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate <br>antibiotic* and grow overnight. *
<br>4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
<br>5.Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol <br>see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
<br>* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically <br>designed to indicate antibiotic resistance.
<br>
<br>Note: There is not enough DNA in each well to perform anything but transformations
<br>
<br> * Transformation:
<br> put your cells+DNA 30min. on ice
<br> incubate them for 2 min at 42 degrees
<br> add 300 microliter LB medium and incubate them at 37 degrees
<br> Plate 250 microliter and 50 microliter
<br> incubate overnight at 37 degrees
<br>
<br>
<br> Searching for protocols
<br> Do a PCR for PhybB on E.coli DH5alpha suspension:
<br> Composition:
<br> Taq 10× buffer: 5μl
<br> dNTPs: 1μl
<br> MgCl2: 3μl
<br> taq 5u/μl: 0.25μl
<br> Forward primer 10mM: 1μl
<br> Reverseprimer 10mM: 1μl
<br> MQ: 38.75μl
<br>
<br>
<br> Analyse them on a 1% agarosegel
<br> PCR conditions:
<br> denaturation: 10min
<br> Cycle 25×
<br>  denaturtaion: 1min
<br>  annealing: 1min (gradient for 60, 65 and 70 degrees)
<br>  extension: 1min
<br> Final extension: 10min
<br> Store infinite




Revision as of 02:40, 1 August 2011

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18-5-11



2PCRs failed yesterday, byproducts
Try to resuspend them better and leave them on ice after pipetting


Searching for protocols
Do a PCR for PhybB on E.coli DH5alpha suspension:
Composition:
Taq 10× buffer: 5μl
dNTPs: 1μl
MgCl2: 3μl
taq 5u/μl: 0.25μl
Forward primer 10mM: 1μl
Reverseprimer 10mM: 1μl
MQ: 38.75μl

Analyse them on a 1% agarosegel
PCR conditions:
denaturation: 10min
Cycle 25×
denaturtaion: 1min
annealing: 1min (gradient for 60, 65 and 70 degrees)
extension: 1min
Final extension: 10min
Store infinite


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