Sequencing

****Sequencing****

 After verifying correct size via colony PCR, growing, and mini prepping, send DNA for sequencing.

**ORDERING**

We use Quintara Biosciences (http://www.quintarabio.com).

Login ID: kmorimckenney@ucdavis.edu

Password: Since this is a public forum- see google docs, primers page

Once logged in, click on the "Order" tab at the top. Then under "Online Order Quick Select" on the left, click the first option, "Online Form".

Type in your template name in the indicated row. For example, "pET28-sfGFP-Dyrk1a-cl.6". Then make sure the type says "plasmid" (assuming you are sending plasmid DNA, don't think we send anything else). Then click the "premixed" option. Lastly, add your primer name (eg. 182R). Each reaction only gets ONE primer. If you need to sequence something with multiple primers, you must add multiple reactions to your order. If you have three primers, you need to send three separate tubes of DNA/primer/water. You don't have to fill anything else out, although, you can if you like. Click "Order" and then "Confirm". Then print the page.

Sequencing is expensive, so please just sequence the Gibson seams first, and if those come back correctly, send any other primers you need in order to fully sequence the gene (I would also recommend sequencing the tag, too, just to be sure). Quintara generally gets sequences back the morning after submission. They pick up Mon-Fri at 4pm. Once you have sequenced everything, add the construct to the plasmid database (☀https://docs.google.com/spreadsheets/d/1iU-kdsn6mdmAw\_TWI7e1ddDmEBz5D-iHHZP8N3ixf5A/edit#gid=0). Once you've done this, and you have a MOM number for your construct, upload the ApE file and all associated sequencing files to the "Ape file database" in the McKenney folder on the MCB server. If you can't access the server, put files on a USB and you can upload them from the iMac on Paige's desk. This is important so that others have access to the file if they will use it, or PCR from it, in the future.

**SAMPLE SUBMISSION**

Total volume should be 15ul. You should aim for 800ng of DNA TOTAL (though anywhere from 700-800 is safe). This means if your DNA concentration is 200ng/ul, you should send 4 ul. If it is 500ng/ul, send 1.6ul (though, 1.5ul would be sufficient). Your ending primer concentration in 15ul should be 1.67uM, so add 2.5ul of your 10uM diluted primer (you can use 0.3ul of 100uM primer, but I stopped doing that when I was getting results back that said "high background"). The rest of the 15ul should be autoclaved DI water.

DNA: (175ng/ul)(Xul)=800ng ---> (175ng)(X)=800ng ---> X=800ng/175ng ---> X=4.57ul ---> round to 4.5ul (787.5ng is close enough)

PRIMER: (10uM)(Xul)=(1.67uM)(15ul)---> X=(25.05/10) ---> X=2.5ul

WATER: (4.5ul)+(2.5ul)+(Xul)=15ul ---> X=8ul water

Label strip PCR tubes with the sample number according to the order you submitted. The first tube needs to have the initials "KOM" and then the sample number "01" and then a horizontal line at the bottom. The rest of the samples only need the sample numbers (eg. "02" and "03" if you have three samples). Tape the tubes sharpie side down (tape will sometimes pull the marker off of the tube) on the printer order page. Place in the designated cold room for pickup (have someone show you, it's too hard to explain which cold room).