Evaluation of DNA probe removal from nylon membrane.
Genetic fingerprinting is one of the most challenging applications of any hybridization membranes. Forensic DNA fingerprinting typically uses samples in the range of 100-400 ng of genomic DNA. To ensure the ability to successfully reprobe the samples, it is imperative that repeated stripping of sample DNA be minimized while stripping of the probe DNA be maximized. By using standard dilutions of K562 cell line, we compared the following three stripping techniques: NaOH at 25 degrees C, formamide (HCONH2) at 65 degrees C and 0.1 x standard saline citrate and 0.1% sodium dodecyl sulfate at 95 degrees C (high-temperature stripping). The largest amount of genomic DNA was stripped from the membrane with NaOH, with the other two techniques removing less. Formamide and high-temperature procedures resulted in a loss of approximately 5-10 ng of DNA per strip. In contrast, the NaOH resulted in a loss of approximately 10-20 ng per strip.
Noppinger
K
K
Broward County Sheriff's Crime Laboratory, DNA Unit, Ft. Lauderdale, FL 33301.
Duncan
G
G
Ferraro
D
D
Watson
S
S
Ban
J
J
D001345:Autoradiography; D016172:DNA Fingerprinting / Q000379:methods*; D015342:DNA Probes / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D005559:Formamides; D006358:Hot Temperature; D006801:Humans; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009757:Nylons; D012150:Polymorphism, Restriction Fragment Length; D012972:Sodium Hydroxide
D015342:DNA Probes; D005559:Formamides; D008567:Membranes, Artificial; D009757:Nylons; C031066:formamide; D012972:Sodium Hydroxide