Arizona_State
Notebook
Amber Mani
Transformation of DH5alpha with PSB1C3 and PSB1A3 backbones
WEDNESDAY, 7/19/2017
In preparation Alyssa and I rehydrated 2 backbones from iGEM, the PSB1C3 and PSB1A3. The C3 was from plate 7 location O,23 and the A3 was from plate 4 location H,2. Will let the plates grow overnight in the incubator and make MP samples, in triplicates of each, tomorrow.
DNA extraction from kit and rehydration protocol:
To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200- 300pg/µL
- With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells
- Pipette 10µL 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. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
- Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
- Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours
- 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.
7/20/2017 - something went wrong with the process, the plates to not show sufficient growth. Tests and plating will be redone today.
Gathered the available colonies and put in LB with (AMP or CHLOR) to grow overnight and replate for more usable growth to be MP. samples in the shaking incu overnight, was able to grab 3 colonies from the A3 and two from the C3. Put everything in the shaking at 345pm
Doing the MP on the A3 and C3 growth tubes. Concentrations:
psb1c3: 80ng/ul
psb1c3: 40ng/ul
psb1a3: 50ng/u
psb1a3: 67ng/ul
psb1a3: 75ng/ul
