III Genomic dna extraction from whole blood 1. Principle Genomic DNA Extraction Kit: In the high salt state, DNA purification resin adsorbed DNA pecificly; while in a state of low-salt or aqueous solution, DNA was eluted down 2. Reagents: purification resin Gn binding buffer washing buffer Purification Column 3. Experimental Steps 1. Add 0. 4ml whole blood to Iml purification resin in a tube. Invert tube 5-6 times gently and leave to incubate for 3 minutes at room temperature. Invert the tube again at the half of the 3 minutes. Spin at 5000 rpm for 3 secs. Discard supernatant 2. Re-suspend pellet in 1 ml of gn binding buffer, Invert the tube. Spin at 5000 rpm for 3 secs 3. Re-suspend pellet in 0.5 ml of washing buffer. Invert the tube. Spin at 5000 rpm for 3 secs Discard supernatant. Repeat this step again 4.The pellet should be white to cream in colour. If pellet is significantly yellow, repeat washing 5. Put the Purification Column in a new 1. 5ml tube, add 100ul ddH20 to the Resin of the Purification Column, incubate for 3 minutes at room temperature, Spin at 12000 rpm for 2 min 6. Finally, the genomic DNA is in the tube IV Polymerase chain reaction PCR: a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNa melting and enzymatic replication of the dna 1. Principle a basic PCr set up requires several components and reagents. These components includ DNA template that contains the DNA region( target) to be amplified Two primers that are complementary to the 3'( three prime) ends of each of the sense and anti-sense strand of the DNA target 2. Preparation l、 Taq PCr Master Mix 、 primers 4、 template dna ddH20 2lul 2X Taq PCR Master Mix 25ulIII Genomic DNA extraction from whole blood 1.Principle Genomic DNA Extraction Kit: In the high salt state, DNA purification resin adsorbed DNA specificly; while in a state of low-salt or aqueous solution, DNA was eluted down. 2. Reagents: purification resin GN binding buffer washing buffer Purification Column ethanol 3. Experimental Steps： 1. Add 0.4ml whole blood to 1ml purification resin in a tube. Invert tube 5-6 times gently and leave to incubate for 3 minutes at room temperature. Invert the tube again at the half of the 3 minutes. Spin at 5000 rpm for 3 secs. Discard supernatant. 2. Re-suspend pellet in 1 ml of GN binding buffer, Invert the tube . Spin at 5000 rpm for 3 secs. Discard supernatant. 3. Re-suspend pellet in 0.5 ml of washing buffer. Invert the tube . Spin at 5000 rpm for 3 secs. Discard supernatant. Repeat this step again. 4.The pellet should be white to cream in colour. If pellet is significantly yellow, repeat washing step again. 5. Put the Purification Column in a new 1.5ml tube, add 100µl ddH2O to the Resin of the Purification Column, incubate for 3 minutes at room temperature, Spin at 12000 rpm for 2 min. 6. Finally, the genomic DNA is in the tube. IV Polymerase chain reaction PCR:a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. 1.Principle A basic PCR set up requires several components and reagents.These components include: DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. 2.Preparation 1、Taq PCR MasterMix 2、primers 3、ddH2O 4、template DNA • ddH2O 21ul • 2X Taq PCR MasterMix 25ul