TABLE 1 |.

Troubleshooting table.

Step	Problem	Possible reason	Solution
1A(iv)	Sample aggregates in large clumps	Sample was dropped too quickly in liquid nitrogen	Maintain maximum liquid nitrogen volume, and introduce sample drops more slowly
1A(v)	Sample is stuck to the inside of the chamber after grinding	Unknown	Scrape off as much sample as possible with a cold spatula. It is not necessary to remove all of the sample
1A(vii) 1B(vi)	After the second centrifugation, the sample is cloudy	Unknown	Sample cloudiness does not necessarily compromise sample quality. Proceed with the protocol, and monitor sample quality and quantity
1A(xv,xvii,xviii), 1B(xiv,xvi,xvii)	Bead volume decreases during washing	Beads may be accidentally aspirated	Avoid the bead bed when aspirating. Keep the needle bore against the side of the tube. Tilt the tube if it helps prevent the removal of beads
1A(xix), 1B(xviii)	Protein did not elute from Ni-NTA beads	Incorrect pH of elution buffer	Ensure that the pH of the elution buffer is correct
1A(xxi), 1B(xx)	No visible bands on the western blot or silver stain	Insufficient material	Samples may need to be loaded at higher quantities, depending on the level of ligase expression. Consider overexpressing the ligase of interest, as this is often the limiting factor for purifying enough material