Revision as of 14:47, 22 June 2006
1) Tell Alain you want to order sequencing reactions. He will set up a purchase order number for you. For the sake of his sanity, try to combine orders as much as possible.
2) For each plasmid + primer combination, combine 8 uL of plasmid with 4 uL of 2 uM primer in one tube of a 8-tube PCR strip.
3) Label each reaction with your initials and a three digit number (i.e. CD001, CD002). Note on your wiki page which reaction is which!
4) Wrap the strip in Parafilm.
5) Go to http://www.genewiz.com. Log in with Alain's email address and a password which I will give you in person (better to not spread it via the interweb). Click "Place DNA Sequencing Order."
6) Enter the number of reactions, and choose "Premix" as the sequencing type and "no" for same day service.
7) Enter the PO Number Alain gave you.
8) For each reaction, enter the label (CD001) and the approximate size of the plasmid in base pairs - you can use the Registry to figure this out. Choose DNA Type "Plasmid" and Primer "Already Added".
9) Submit the form, print out two copies. Keep one for your own reference and give the other to Alain along with the wrapped tubes.
We have a box in the freezer in the large room labeled "20 uM Primers." The originals are kept somewhere else; if more 20 uM stock is needed, ask Chris and he'll pull them out.
Since the working concentration is 2 uM, you should dilute the primer 1:10 before adding it to the reaction tube. You may want to make a large batch of 2 uM primer for individual use in the future; I made 200 uL of both VF2 and VR, so there's enough for each person to make their own stock of 20 uL primer + 180 uL dH20. In general having your own stock of commonly used reagents is a good idea to avoid accidental contamination - when everyone is going into the same tube all the time, mistakes are bound to happen and they'll affect everybody!