Sequencing of psb1c3+T7 promoter+silk biobrick'

The miniprepped samples from 7/29 were sent in for sequencing.

Running HoneyBee Protein on SDS PAGE gel

In the morning I incubated the purified inclusion bodies in a slightly above RT water bath in order to resolubilize the SDS and the protein.
I then took a 40 ul fraction and put the rest in the 4c room for storage.
Fasih Asahn taught me how to load a pre-cast SDS PAGE gel with my samples.

Put the samples on ice.
Establish what sample will go in which lane.
- From left to right it is F1,S1,F2,F3,S2,S3, Final product, and BSA positive control.
Calculate and prepare the Sample Buffer (2x).
- The sample buffer is composed of 5% Beta-mercapto ethanol, and 95% Laemli sample buffer.
- Since I have 8 samples, 40 ul each, I will need 320 ul of this 2X sample buffer.
Add the BME in the fume hood into an epi tube, and add the Laemli buffer. (must be prepared fresh every time.)
Add 40 ul of this prepared Sample buffer to each of the 40 ul samples and mix thoroughly by pipetting.
Set the heat block to 95C and incubate tubes for 5 minutes.
While the heat block is warming up, set up the gel rig.
Prepare the 10X running buffer, lock in the apparatus, shake up the buffer, and fill up the inner chamber before adding to the outer chamber.
Once samples have been boiled, remove the comb from the gel, and add samples to the lanes.
- Don't forget to add ladder.
Set the voltage at 200V and run approximately 40 min, or until the dye front hits the black line on the Bio Rad apparatus.
- I noticed that the dye front on the lanes for the positive control and the lane with the final product ran significantly slower than the other lanes. Vinson says that this could be due to higher protein concentration in those lanes.