Template:SBB-Protocols AgaroseGel

Running an Agarose Gel

• The gels are pre-made with Sybr Green I DNA stain and a 1% agarose concentration.
• There are 10 wells. Each one holds about 20 uL max.

1. Sign up for your lanes on the sign-up sheet.
2. Take a small amount of your sample (can be as low as 4-6 uL) and add loading dye to it. The loading dye is 6X, so you should use at least 2 uL of dye with 10 uL of sample - but using a greater ratio of loading dye is fine (such as 6 uL of sample and 2uL of dye).
3. Pipet your mixed sample into the well.
4. The last person to load their sample into the gel also:
   1. Loads the ladder
   2. Starts / stops the gel
   3. Images the gel
   4. Posts the image on the wiki along with the table of who signed up
5. Load about 10 uL of the 1:5 diluted 2-log ladder (the 1:5 dilution is labeled "Test Ladder").
6. Connect the gel box. Be sure to check that the gel faces with the samples on the negative (black) side.
7. Run the gel at 200V. Stop it when the lower dye band goes 1/2 to 2/3 of the way down the gel.
8. Imaging:
   1. Be sure that both the camera and the imaging box are on. There are two switches, one on the black power supply behind the imaging box, and the other on the imaging box itself.
   2. Start Quantity One (shortcut is on the desktop, it's the red icon)
   3. File --> Chemidoc XRS
   4. Select live preview
   5. Turn on the EPI-white light on the box
   6. Position the gel in the box
   7. Close the box
   8. Switch on the Trans UV, turn off the white light
   9. Looks good? Click freeze.
   10. Click auto expose
   11. Save the picture to 140L-Spring 2012 (folder is inside My Documents). Save it as 2012_MM_DD_gel#_ssb2012spring.1sc where MM = month (02 for Feb) DD = day and # = gel number for that day
   12. Export as JPEG, Quality = 100. Save as 2012_MM_DD_gel#_ssb2012spring.jpg (like above, but with .jpg instead of .1sc)
   13. Upload to the wiki
9. Uploading to the wiki:
   1. Go to the gel images page SBB12_gels
   2. Copy this: (change the appropriate stuff: #, MM, DD, and entries)

===Class Gel #===
[[Image:NEB_2-log_ladder.gif]][[Image:2012_MM_DD_gel#_ssb2012spring.jpg]]<br>

Lanes:<br>
{| border="1" cellpadding="2"
!width="1"|1
!width="1"|2
!width="1"|3
!width="1"|4
!width="1"|5
!width="1"|6
!width="1"|7
!width="1"|8
!width="1"|9
!width="1"|10
|-
||entry one||two||three||four||five||six||seven||eight||nine||ten
|}

• Click the red link and upload the picture! Done!