02-06-11
Dissolved the rest of the primers needed in this project.
PCR hyBB with taq DNA polymerase. Testing strain E.coli TG1 and DH5alpha.
for E.coli TG1: isolate DNA with magnetic beads of the 7th floor.
Do PCR with the isolated DNA (concentration is very low, one co worker on the 7th floor told me to use 10 to 12 microliter of the DNA and then it should work! (at least it always worked for her))
Taq 10× buffer: 5μl
dNTPs: 1μl
MgCl2: 3μl
taq 5u/μl: 0.25μl
Forward primer 10mM: 1μl
Reverseprimer 10mM: 1μl
template: 1μl, but 12 μl for TG1 sample!
MQ: 38.75μl (but for sample TG1 use 26.75μl MQ)
PCR conditions:
denaturation: 10min
Cycle 35×
denaturtaion: 30s
annealing: 30s (gradient for 60, 65 and 70 degrees)
extension: 1min
Final extension: 10min
Store infinite at 4 degrees
Analyse them on a 1% agarosegel
PhybB has correct size!
Can use it for cloning, since taq polymerase makes 1 mistake per 1000bp according to Martijn, and PhybB plus prefix and suffix
is around 450bp.
Use some samples of the biobricks for cloning:
Transforming biobricks we need
Protocols for transforming biobricks in cells on http://partsregistry.org/Help:Spring_2011_DNA_distribution
DNA Kit Plate Instructions
To use the DNA in the Distribution Kit you may follow these instructions:
1.With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you
want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to
cross contamination between the wells.
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
the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
3.Transform 1 or 2uL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate
antibiotic* and grow overnight. *
4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5.Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol
see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically
designed to indicate antibiotic resistance.
Note: There is not enough DNA in each well to perform anything but transformations
* Transformation:
put your cells+DNA 30min. on ice
incubate them for 2 min at 42 degrees
add 300 microliter LB medium and incubate them at 37 degrees
Plate 250 microliter and 50 microliter
incubate overnight at 37 degrees
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