STEP	PROBLEM	POSSIBLE REASON	POSSIBLE SOLUTION
50 (gDNA Isolation)	Lysed material is unable to be forced through 23G needle.	Cell pellet has not been lysed small enough during previous steps.	Forced lysed material through 20G needle until the solution passes easily through and then switch to a 23G needle until the solution passes easily through. Move on to next step in protocol.
54 (gDNA Isolation)	Sample mixture has not sufficiently flowed through mini-spin column allowing for collection of filtrate.	Sample mixture may be too thick to flow through the mini-spin column therefore blocking the collection of filtrate into the collection tube.	Centrifuge sample again at 8000 x g for 1.5 minutes. Place the mini-spin column in a clean collection tube. Discard the tube containing the filtrate. Continue to next step in protocol.
60 (gDNA Isolation)	gDNA concentration of sample is too low for HLA Typing.	The cell pellet used for isolation of gDNA contained less than 2x107 CD34neg cells (UCB samples) or thymocytes (Thymus samples).	Redo gDNA isolation using cell pellet containing more than 2x107 CD34neg cells (UCB samples) or thymocytes (Thymus samples).
68 (HLA Typing)	Multi-channel pipette tips come into contact with the bottom of the wells.	Sample mixture was not pipetted to the side of the wells.	Change all pipette tips to avoid contamination of primers and further invalidation of results.
68 (HLA Typing)	Multi-channel pipette tips are drawing up different amounts of sample mixture.	Pipette tips are not filling properly due to reasons such as air bubbles being drawn into certain channels, pipette tips not fitting well, or insufficient pipetting technique.	Repeat sample mixture draws until volumes look correct in pipette tips. If this does not work, change all pipette tips and continue to repeat sample mixture draws until volumes look correct in pipette tips.
72 (HLA Typing)	Evaporation of sample mixture from all or some wells in PCR tray leading to dropout wells in HLA typing analysis.	Plate was not sufficiently sealed and may have contained either air pockets within the center of the tray or puckering of the edge of the optical tray seal.	Repeat HLA typing protocol for sample and ensure PCR tray is sufficiently sealed before placing into PCR machine. Use a seal applicator to press seal over the top of all the wells and along each edge of the PCR tray. Be careful to make sure there is no puckering of the plate seal at each corner of the tray. Ensure there are no air bubbles formed near the raised letters and numbers along the top and left edges of the PCR tray.
75 (HLA Typing)	SureTyper program does not automatically call locus instead flagging it as "ambiguous rare”	More than one genotype can explain the reaction pattern so the program is unable to automatically call the locus.	Ambiguous rare test results may be manually selected via allowing SureTyper program to choose the only valid test call that does not require the assignment of a rare allele. Practice on Supplementary Data 1 data set with Supplementary Data 2 to guide what the finished product should look like.