Troubleshooting.

Step	Problem	Possible reason	Possible solution
73	Inefficient co-purification of RNC complexes upon affinity purification.	Factor-RNC complexes are not stabilized enough to sustain the purification process.	Fast ATP depletion (for Ssb) or chemical crosslinking. In vitro ATP depletion or crosslinking can be accelerated by the stepwise thawing of small frozen lysate amounts in the stabilizing buffer (that provides ATP depleting conditions or contains chemical crosslinker). Add new powder only after the previously added powder is completely melted.
73	Factor-independent binding of ribosomes during affinity purification in the control (high background).	Inefficient washing condition.	High background binding may be reduced by applying harsher washing conditions, either by changing the composition of washing buffer (e.g. by increasing the salt or detergent concentrations), or by washing more intensely (more or longer washing steps). Another possibility is to change the kind of beads used for the purification.
87	Gel pieces remain in the gel breaker tube after centrifugation.	The gel pieces are too big.	Take a sterile 200-µL filter-tip and smash gel-pieces, centrifuge again.
90	The 1,000 µL filter-tip clogs while transferring the broken gel pieces	The gel pieces produced during the gel breaker tube centrifugation are too big.	Cut the pipet tip to widen the tip opening.
118	Less than 5 pmol of RNA are available for the linker ligation reaction	Small amount of RNC- factor complexes used for acid phenol extraction of nucleic acids	If only small amounts of extracted RNA fragments are available, the amount of linker 3-L1 and Linker L(rt) should be reduced to maintain the proper stoichiometry of reactants. We have successfully generated libraries starting from 0.5 pmol of dephosphorylated RNA by using 10-times less linker 3-L1 and Linker L(rt).
140	Inefficient ligation reaction	The ligation reaction (and other enzymatic reactions) may be inhibited by elevated salt concentration.	The amount of co-precipitating salt can be reduced by precipitating nucleic acids over night at −20 °C instead of precipitating one hour or more at −80 °C.
227	Limited amount of cDNA product obtained by PCR	Small amount of PCR template.	Increase the number of PCR cycles. Be aware that as little cycles as possible should be used to limit the PCR bias and to avoid the accumulation of unspecific PCR-products.
235	Elevated expression of stress genes	Cells were exposed to stress (e.g. nutrient starvation) during growth or cell harvest.	Cell harvest by filtration should be done using prewarmed equipment and as fast as possible. Cells must be scraped and frozen immediately once the medium has passed the membrane.