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TROUBLESHOOTING GUIDE
 

FosPrep™ 96 Fosmid Prep Kit

OBSERVATION
POSSIBLE CAUSE
RECOMMENDATIONS

Poor DNA yield.

 Poor cell lysis due to large amount of
bacterial cells.		 dot Use less rich media like 2xYT.
dot Use static growth culture conditions.
dot Decrease incubation time.
Poor cell lysis due to inactivation of
enzymes in Lysis Solution/Enzyme
Mix.		 dot Store Enzyme Mix at -20°C.
dot Store Lysis Solution/ Enzyme Mix at 4°C.
Combined solution is stable for two weeks.
dot Prepare fresh Lysis Solution/Enzyme Mix.
Failure to resuspend bacterial
cells completely.		 Increase vortexing or mixing time until pellet is
completely dispersed.
Fosmid DNA was not induced to high
copy.		 dot Include the inducer when growing cells for
fosmid preparation.
dot Maintain un-induced culture or glycerol
stocks in the presence of glucose.
dot Ensure appropriate antibiotic was included
during all stages of growth.
Cell pellet too tight. Decrease centrifugation force or time.
Failure to pellet cells. Increase centrifugation time.
Over-drying of DNA pellet. dot Decrease drying time.
dot Increase the incubation time or temperature for elution.
Ethanol used instead of Isopropanol. Use Isopropanol as suggested.
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OBSERVATION
POSSIBLE CAUSE
RECOMMENDATIONS

Degradation seen in
restriction analysis.

 Poor sample prep. Follow recommended protocols carefully.
Incomplete removal of lysate and washes. Use inverted spin to remove lysate and washes.
OBSERVATION
POSSIBLE CAUSE
RECOMMENDATIONS

Poor sequencing results.

 Poor sample prep. Follow recommended protocols carefully.
  Too much or too little DNA used. Adjust the DNA input based on the BigDye®
volume and total reaction volume.
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protocols Diagrams and Protocols

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