For Yeast, Use Blood & Tissue Genomic DNA Extraction System. For Filamentous Fungi And Mushroom, Use Plant Genomic DNA Extraction System. Yield of genomic DNA extracted is low. Several Things To Pay Attention To: 1) The Sample May Not Contain Enough Amount Of Genomic DNA. Increase The Sample Amount, Proteinase K, And Buffers Proportionally. 2) Elution Buffer Preheated At 70°C Is Used. 3) DdH2O, Tris-HCl, Or TE Used Should Be Of PH 8.0-9.0. 4) After Adding Elution Buffer Into The Column, Stand The Column For 1-5 Minutes Before Centrifuging To Collect Eluted DNA.

Genomic DNA Extracted From Paraffin-Embedded Tissue Is Usually Degraded. It Does Not Show As A Clear Strong Band Of About 20-30 Kb As We Usually See When Fresh Sample Is Used. It Is Because Genomic DNA In Paraffin-Embedded Tissue Unavoidably Suffers From Degradation When Sample Has Been Treated And Stored Over A Long Period. DNA In This Case Is Not Suitable For Southern Blotting And Restriction Analysis Due To The Smearing. However, It Is Applicable For PCR.

If A Sample Is Rich In Protein, E.G. Fish Flesh, Complete Digestion Will Not Be Achieved Using The Amount Of Proteinase K And Buffer Suggested In The Protocol. If A Sample Cannot Be Digested Completely Or Appears Very Viscous, Add More LYS Buffer And Repeat Incubation. Centrifuge The Sample At Full Speed For 5 Minutes To Remove Undigested Remains And Only Use The Supernatant In The Following Steps. In The Subsequent Preparations, A Lower Amount Of The Sample Should Be Used. A General Rule Of Thumb Is To Start With Half Of The Maximum Amount Of Sample Suggested. When There Is No Problem In Digesting The Sample Completely And Passing The Lysate Through The Column, Amount Of The Sample To Be Applied Can Be Increased Gradually In The Next Preparations.

The Key Is To Use Fresh Sample And Not To Overload The Column. Low Yield Or Purity Of Genomic DNA Is Usually Due To Incomplete Digestion Or Incomplete Lysis Of The Sample. Starting With A Maximum Amount Or Volume Of Samples Does NOT Usually Give The Best Yield Of DNA. On The Contrary, It Usually Results In Incomplete Sample Lysis And Degradation Of Proteins, Thus Making Extraction Of All DNA From The Sample Unfeasible. Further, It Always Requires Subsequent Removal Of Undigested Residues And Yields Viscous Sample Lysate. When The Lysate Is Too Viscous, It Not Only Has Difficulty In Passing The Column, But Also Indicates The Presence Of An Abundant Amount Of Contaminants Such As Proteins And Salts. Contaminants Of High Amount Not Only Affect DNA Binding, But Also May Not Be Washed Off Completely, Leading To Carry Over To The Eluted Genomic DNA. Therefore, A Good Quality And Yield Of DNA Is Only Expected When A Sample Is Completely Digested. We Advise Starting With Half Of The Maximum Amount Of Sample Suggested. When There Is No Problem In Digesting The Sample Completely And Passing The Lysate Through The Column, Amount Of The Sample To Be Applied Can Be Increased Gradually In The Subsequent Preparations.

This Indicates That RBC Or Hemoglobins Have Not Been Completely Lysed Or Digested. During Incubation For Proteinase K Digestion, The Sample Should Be Mixed Every 3-5 Minutes By Vortexing And Inverting To Completely Disperse Proteinase K And Samples.

Several Points Should Be Noted To Avoid DNA Degradation: 1) DNA Degradation Occurs When The Sample Is Not Fresh Or Is Stored Improperly For A Long Time. Samples Not Used Immediately Should Be Flash Frozen In Liquid Nitrogen And Stored At -80°C. Genomic DNA In Samples Stored At Room Temperature, 4°C, Or -20°C Are Subjected To Degradation. It Is Also Not Advised To Keep Samples In Buffer Or Medium And Stored At -80°C. 2) For Whole Blood Samples, If They Are Stored At Room Temperature For More Than 2 Days Or At 4°C Or -20°C, Genomic DNA Isolated Appears Smearing At An Extent Proportional To The Storage Time. 3) Use Fresh TAE Or TBE Running Buffer For Electrophoresis, Repeatedly Used Running Buffer May Be Contaminated With DNase. 4) If Isolated DNA Needs To Be Stored For A Long Time, Use 10 MM Tris-HCl (PH 9.0) Or TE For Elution. DdH2O Is Not Advised In This Cause Because DNA Fragments In H2O Suffer From Gradual Degradation Through Acid Hydrolysis Readily. 5) If DNA Is To Be Used Frequently, Elute In 10 MM Tris-HCl (PH 9.0) Or TE And Store At 4°C. Keep DNA At -20°C Only For Long-Term Storage. Repeated Freeze-Thaw Cycles Can Cause Shearing Of Genomic DNA. 6) Genomic DNA Extracted From Paraffin-Embedded Tissue Is Usually Degraded. It Is Because Genomic DNA In Paraffin-Embedded Tissue Unavoidably Suffers From Degradation When Sample Was Treated And Stored For A Long Time. DNA In This Case Is Not Suitable For Southern Blotting And Restriction Analysis Due To The Smearing. However, It Is Applicable For PCR.

If It Is Blood Stain (Dried Blood) On A Piece Of Filter Paper, It Is Still OK To Use Our Blood & Tissue Genomic DNA Extraction Miniprep System To Extract DNA From It. What Dr. K. Nobuto Can Do Is To Cut The Filter Paper Into Small Pieces (About 10 Mm2). Place 1 To 4 Pieces (Depends On How Concentrated The Blood Stain Is) Into A Clean 1.5-Ml Eppendorf Tube. Add 20 Ml Proteinase K And 200 Ml LYS Buffer Into The Sample. Mix Immediately ByVortexing For 20 Seconds. Follow The Tissue Protocol From Step 3.