iPCR_Jill - INVERSE PCR (Jill) 1. Digest 2.5g of genomic...

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INVERSE PCR (Jill) 1. Digest 2.5 µ g of genomic DNA with restriction enzymes. Stop reaction at 65˚C for 20’. 2. Fractionate on a 1% agarose gel to check DNA has been cut. 3. Set up self-ligation reaction: 40 µ l 10 x DNA Ligase buffer 1.5 µ l NEB concentrated DNA ligase 358.5 µ l 1 μ g digested DNA plus dH 2 O Incubate overnight at room temperature. Do not put in more that 1 µ g of DNA as fragments may ligate to each other rather than to themselves. 4. Add 400 μ l 25:24:1 phenol:chloroform: IAA. Vortex and spin for 5’ in microfuge. Remove aqueous layer to fresh tube. 5. Add 2.5 volumes ethanol and 0.1 volume 3M NaOAc pH4.5. Precipitate at –20 o C overnight or at –80 o C for 1 hr. 6. Spin in microfuge for 10’ at full speed. Wash pellet with 70% ethanol, spin again and drain off supernate.
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This note was uploaded on 08/01/2009 for the course HORT hor-11-12 taught by Professor Park during the Spring '09 term at A.T. Still University.

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iPCR_Jill - INVERSE PCR (Jill) 1. Digest 2.5g of genomic...

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