In our lab, we frequently screen 50+ clones to screen for incorporation of a tag after CRISPR. To do this, we take cell pellets from clones growing at the 24-well stage, lyse in a minimal volume of 2x Lamelli's buffer neat or mixed 1:1 with Adam's buffer (50mM Tris pH8.1, 100mM NaCl, 1%SDS, 15% glycerol). Most of the time we snap freeze the pellets in LN2 the day before and thaw them for lysis later. I vortex these pellets for ~10s each in the lysis buffer, then heat at 90C for 3mins while vortexing for 5s right before heating, after 1:30 of heating, and again at the end of the 3mins. In my hands, the vortexing seems to be necessary for reducing how goopy the sample is. However, I know that other people in my lab heat for much longer without vortexing, or do some other combination of vortexing and heating.
My main issue is that sometimes, after blotting these samples for whatever protein I'm looking for, I end up with a ton of extra bands below WT, making me think that whatever I'm doing is causing degradation of the protein leading to extra bands. These bands tend to be identical between samples. The only explanation I can think of is that maybe lysing in too small a volume makes it more likely to have protein degradation but my PI doesn't think that any of our lysis methods would have this effect and cause banding.
Does anyone have any recommendations for how to lyse a fairly large number of samples at the same time? Thanks in advance for your help!
Lincoln, you could try to first resuspend the cells in a little cold PBS (or similar) before adding the buffer that contains the SDS. Might speed things up.
Extra bands very likely are due to some protease activity, thus a little EDTA and PMSF (and possibly other protease inhibitors) added to the resuspension buffer might do the trick.
- Badges
- Science topic
- Similar topics
- Cognitive Science
- Thinking