Problem | Solution |
---|---|
Slow Growing 293T | This is often resulting from high passage number. Try to use 293T cells that are below passage 12. |
DNA Precipitation | If the DNA is not pure it may cause excessive precipitation during the transfection set up. The Nucleobond Xtra Maxi EF kit is recommended for endotoxin removal, and thorough rinsing of the DNA precipitate with 70% ethanol can remove excess salt. |
Cell Clumping | If the trypsinization step was insufficient to dissociate the cells from one another, it is recommended to rinse again with trypsin to improve recovery. |
Uneven Cell Plating | This may occur if the incubator shelf is not level. |
Cells Peeling Off | This can occur for several reasons: too many cells were plated, or the medium changes were performed too vigorously, or the HYPERflask was repeatedly knocked. |
Column Integrity Fail | This is very rare but when it does occur the column must be replaced. |
Flow Path Leak | Check all the connections throughout the flow path as this is usually caused by a single loose connection. |
Blocked filter | If too much particulate matter or protein (serum) is in the LV-containing medium then the filter may start to block. Reduce the protein content in the LV-containing medium by using serum-free medium. If serum-free medium was used and the permeate flow seems slow then try closing the permeate to increase the internal pressure in the filter to try unblocking some of the pores in the membrane. |
High inlet pressure | This often occurs as the filter is beginning to clog. Reduce the back pressure (if additional back pressure has been applied) and reduce the flow rate. |
Aggregation | If too much protein is removed then there is a substantial increase in aggregation. Try to keep some protein present in the diafiltration mix and avoid diafiltrating in pure DPBS. |
Overheating | FP2 can heat up during use, threatening the stability of the vector. As a precaution, keep the reservoir tube on ice during the second concentration stage. |