Problem loading DNA / agarose gel - (Apr/21/2011 )

Dr_Watson on Fri Apr 22 13:19:05 2011 said:

thank you all for your replies and if you think of anything else, just let me know

have a happy easter, or Bonnes paques or whatever good weekend!!

So latest news, I've made a new gel concentrating on the buffer. Which means, I used the same amount of DNA, same loading dye, not even bothering to centrifuge the samples this time. So as I said I focused on the buffer, adding at least 300mL of buffer on top of the gel and this time everything was ok. DNA stayed in the wells.

so just letting you know

Sorry for the late response to this thread. I had this same issue after doing my maxi prep or mini prep. After I would finish my prep I would try to load the DNA into the well and it would float from the pipette up out of the well as it loaded. In order to get around this I had to DILUTE my DNA sample. Yes, I know this seems backwards but this was suggested by my PI and it worked.

i would check the 260nm/230nm ratio on a nanodrop, if really off - say below 1.8 -  i'd say there is some residual contamination from your plasmid prep. If you are using  a spin column kit , such contamination may originate from the washbuffer which contanis usually ethanol, and that may cause the sample to "blow out" of the well. There usually is a drying step in these kit protocols where you spin the column empty for 1 min, you may try to extend this step to 2 min, or incubate the empty column a few minutes at room temperature on the bench to get rid of residual ethanol. Also you may use ddH20 for elution istead of the provided elution buffer.

This is caused by ethanol in your sample. You need to remember to do the final high speed dry spin of your columns after emptying the collection tube. The reason diluting your sample works is that it reduces the percentage of ethanol.