Methods

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Staining a SDS-PAGE gel with Coomassie Blue

1) Cover the gel with coomassie staining solution and leave on a shaker at slow speed for 30 minutes
2) Remove the coomassie stain (can be reused), and replace with destaining solution for 30 minutes
3) Remove the destaining solution (into waste bottle) and replace with destaining solution for 30 minutes-overnight
4) If desired, repeat the destaining process
5) When finished destaining, leave the gel in water. If desired preserve the gel in cellophane.



Staining an agarose gel with ethidium bromide

1) After electrophoresis place the gel into the staining solution for 15 minutes with mild shaking
2) Place the gel into water and visualize under a UV lamp



Running whole E. coli cells on an SDS-PAGE gel

1) Pellet 500 μL of culture in a micro centrifuge tube at maximum speed for 2 minutes
2) Remove the supernatant and add 50-250μL of 1x loading buffer with 50mM Dithiothreitol to the samples, depending on the size of the pellet.
3) Resuspend the pellet in the loading buffer, boil for 10 minutes
4) Place the microcentrifuge tubes on ice for a minute, then centrifuge at maximum speed for 2 minutes
5) Load 5μL from the top of the sample to your SDS-PAGE gel and enjoy.



Computer setups for parallel computing for ws2 and ws3 only

1) Make sure that you have THE SAME USER on both computers
2) Generate keys to ssh from ws2 to ws3 and ws3 to ws2 without passwords on each workstation with the command ssh-keygen -t rsa
3) On ws2, go to ~/.ssh folder. scp id_rsa.pub (the public key) to ws3:~/.ssh/ but rename to authorized_keys--> VERY IMPORTANT
4) Do the same to ws3. if confused, read http://pkeck.myweb.uga.edu/ssh/
5) Now you should be able to ssh ws2>ws3 and ws3>ws2 without passwords.
6) Now you need to copy the id_rsa.pub of ws2 into its own authorized_keys file underneath ws3's id_rsa.pub key so that you can ssh into yourself
from the root user. Also do the same for ws3.
7) Now copy your files to the /xplor-share/ root folder. It should appear on both workstation if the keys worked right
8) chmod 777 all your directories and files (command ls -l: should see -rwxrwxrwx in front of required files)
9) type in the command line "xplor -py annealScript.py -parallel -machines machines.lst -o log
the machines.lst file contains a list of the cores on the computers. ws2=12cores and ws3=6cores so 16 cores max at a given time.



Using the LSQKAB script

1. Install CCP4 to /opt/CCP4 (requires root)
2a. If you use the TCSH/CSH shell: edit ~/.cshrc to include "source /opt/CCP4/ccp4-6.2.0/include/ccp4.setup-csh" and commit "source ~/.cshrc" to the terminal.
2b. If you use the BASH shell: edit ~/.bashrc to include "source /opt/CCP4/ccp4-6.2.0/include/ccp4.sh" and commit "source ~/.bashrc" to the terminal.

This will allow CCP4 software, including LSQKAB, from your user.

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