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        HLA Typing for A2.1 Transgenic Mice, protocol

        互联网

        1005

         

        DNA Digestion and Extraction from Mouse Tail

        v Cut about 5mm of mouse tail, place in a 1.6mL eppendorf tube on ice (if very cold, the tail is not going to stick to the teflon pestle)
        v Homogenize tails with sterile teflon pestle thoroughly
        v Add 100uL (or 200ul) Batteys digestion buffer (0.1M NaCl, 0.2M sucrose, 0.01M EDTA, 0.03M Tris, pH 8.0, and 0.4-1% SDS)
        v Add 5uL(or 10ul) proteinase K (20mg/mL stock)
        v Incubate at 55?C one hour, vortex every 15min
        v Add 17.5uL(or 35ul) of 8.0M potassium acetate, mix by vortexing, incubate on ice for one hour.
        v After incubation, spin down in cold room (@14k rpm 5-10min) and carefully remove the supernatant and transfer to a new tube.

        ~undefinedDo the following steps under the fume hood!!!!! *

        Add 1 volume phenol and 1 volume of chloroform, vortex and spin @14k rpm 5min
        Take the upper phase, transfer to a new tube, add 1volume of chloroform, vortex and spin @ 14k rpm 5min
        Transfer supernatant into new tube
        Add 2.5 volumes freezer cold 100% ETOH (flick tube with finger to get the DNA to precipitate)
        Spin @14k rpm 20 min in cold room (@ 4?C), discard supernatant carefully (dont dislodge the pellet!!)
        Rinse pellet with 2.5.volumes of freezer cold 70% ETOH (finger flick to mix, spin down in cold room and discard supernatant)
        Let pellets air-dry 15-20min
        Add 30-60uL sterile water (or TE buffer) to dissolve DNA (pipet up and down to dissolve DNA).


        Items needed:

        Digestion buffer: (0.1M NaCl, 0.2M sucrose, 0.01M EDTA, 0.03M Tris, pH 8.0, and 0.4-1% SDS)
        Proteinase K: 20mg/mL stock
        Phenol
        Chloroform
        8M Potassium Acetate
        100% ETOH (stored in -20°C freezer)
        70% ETOH (stored in -20°C freezer)
        Sterile water (or TE buffer)

        PCR of the HLA-A02 gene: ref Krausa et al , Tissue Antigens 1995: 45 , 223-231.

        1)1st PCR: amplification product is 813bp.

        primers AL#37: CCT CGT CCC CAG GCT CT
        primer AL#AW: TGG CCC CTG GTA CCC GT

        DNA preparation: quantify DNA by OD reading and prepare 1ug/10ul for PCR reaction.

        For 1 reaction: 50ul

        10x Buffer 5ul
        dNTP's 4ul
        MgCL2 4ul
        Primers 2.5ul each
        TaqPol 0.5ul
        H2O 21.5ul
        DNA 1ug in 10ul H2O

        Stock solutions:

        10x buffer: 670mM Tris base pH 8.8, 166mM ammonium sulfate, 1% Tween 20
        dNTP's: mix volume/volume each of the 10mM dNTP. Final conc in PCR mix is 200uM each.
        MgCl2: stock is 25mM
        Primers: stock is 20uM

        PCR cycles: program#139 in C209D.

        1cycle: 94°C 3min
        5cycles: 96°C 25sec
        70°C 45sec
        72°C 30sec
        21cycles 96°C 25sec
        65°C 45sec
        72°C 30sec
        4cycles 96°C 25sec
        55°C 1min
        72°C 2min
        1cycle 72°C 10min
        1cycle 4°C forever

        Run 10ul of the product on a 1% agarose gel.

         

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