Molecular Biology Pro Tip-If you gel purify DNA and your subcloning is not working, your UV box is probably frying your DNA--switch to blue light.
A colleague's lab's subcloning stopped working suddenly and they couldn't figure it out. My lab's subcloning works fine.
We figured out that when our 13+yo communal gel doc died they switched to a newer system that fried their DNA. I avoid gel purification like I avoid Covid so my lab was fine. We finally figured it out 3 mo later.
#Science
@MCDuncanLab good to know!
As someone who has gel purified because “that’s the thing you do,” how do you avoid that step? Make sure the PCR is clean, or just pick a few more colonies?
DPNI only cleaves methylated or hemi methylated DNA so your PCR is safe but your template is toast!!
@askennard
DpnI digest your PCR to remove template.
PPTase vector and if possible digest with a third enzyme to prevent religation of excised MCS.