MATERIAL REQUEST / FAQ
Deisseroth Lab/Optogenetics Innovation Lab
Students, postdocs, lab managers, researchers: For all Deisseroth Lab generated materials, including MTAs for vector cores, please ask your PI to send an email to Dr. Karl Deisseroth and Charu Ramakrishnan (firstname.lastname@example.org, email@example.com) describing the materials you need.
So that we can send quickly, please ensure the following information is sent in the email by the PI:
- Name and address of the institution
- If a signatory from your institution who is other than the PI needs to sign the MTA, the name and address (physical and electronic) of that authority
- Exact name of the construct/virus you want to order. If you are unsure which one you will need or may need others in the future, we can provide you with an MTA that has all 100+ constructs currently deposited with the vector cores.
FAQ for ordering materials from the Deisseroth Lab
- What do you share?
- Virus (in some cases) in a collaborative mode
- MTAs for all the following vector cores:
- How do I know what to ask for?
- How do I ask for materials?
- For DNA requests, please ask your PI to send an email to Dr. Karl Deisseroth and Charu Ramakrishnan (firstname.lastname@example.org, email@example.com) describing the materials you need. Please make sure to include all relevant shipping information
- For information please see #2c above
- For viruses, please ask your PI to send an email to Dr. Karl Deisseroth and Charu Ramakrishnan (firstname.lastname@example.org, email@example.com) describing the materials you need and we will send the lab PI the MTA that can be used for all subsequent orders of the same virus. Every new virus needs a new MTA.
- What do I do with the DNA?
Please store the DNA at -20 C. All of the electronic information for the probes (sequences, maps, protocols, and strategies) is on a dedicated website for convenience: http://www.optogenetics.org.
Experimental details on in vivo devices and other aspects of use can also be found the website. All of the plasmids carry an Ampicillin resistance gene. For the lentiviral and AAV plasmids, please be sure to use a recombination deficient strain of bacteria such as Invitrogen's OneShot Stbl3 cells to avoid unwanted recombination.
Please use 0.5-1.0 ul of DNA to transform bacteria and plate out transformants on LB + agar plates containing 50ul/ml Ampicillin. Use a single colony to inoculate 250-500 ml LB containing 50ul/ml Ampicillin. Purify DNA from bacterial culture using Qiagen Endofree maxi or mega kit. Confirm your DNA by sequencing if possible.
- What do I do with the viruses?
Please store the viruses at -80 C. All the vials from UNC Vector Core contain 100 ul of virus. When you first want to use viruses from a 100 ul aliquot, please thaw on ice for 20 minutes, re-aliquot as 5-10 ul and refreeze at -80 C. AAV can withstand a couple of freeze-thaw cycles. Once you have thawed a smaller aliquot (5-10 ul), you can use 0.5 to 1 ul/injection site (depending on the viral titer) and store any unused virus at 4 C for upto a week.
- Which serotype of AAV do I use?
The most commonly used serotype of AAV is 5. The spread of the virus in the brain tissue increases from AAV2 to 5 to 8 to 9. It will be at the discretion of the user, depending on the brain area and cells targeted, the amount of spread needed, and the time of expression desired, which serotype to use. AAVdj is a newer virus serotype exclusively being packaged at the Stanford Vector Core.