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        常用试剂

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        1228

        一 细菌培养试剂 - 返回 -
        LB培养基

           NaCl            10 g
           Yeast extract        5 g
           Peptone           10 g
           Add dH2O to          1000 ml
           Aliquot to 500ml flasks (250 ml per flask). Seal with sealfilm
           and autoclave them. Store at room temperature.

        LA固体培养基

           NaCl            10 g
           Yeast extract        5 g
           Peptone           10 g
           Agar powder            13 ~ 15 g
           Add dH2O to          1000 ml
           Aliquot to 500 ml flasks (250 ml per flask). Seal with sealfilm
           and autoclave them. Store at room temperature.

        X-gal (20mg/ml) :

           20mg X-gal 溶于 1ml 二甲基甲酰胺中, -20 ℃避光保存;

        IPTG (200mg/ml) :

           1g IPTG 溶于 4ml 去离子双蒸水中,定容至 5ml ,过滤灭菌后 -20 ℃保存;

        Amp ( 100mg/ml ):

           1g Amp 溶于 4ml 去离子灭菌双蒸水中,定容至 5ml , -20 ℃保存



        二 质粒抽提试剂 - 返回 -
        Solution Ι
        Cell resuspension solution (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, RNase A 100 μg/ml)
            1 M Tris-HCl (pH 7.6)             2.5 ml
            0.25 M EDTA                   2.0 ml
            ddH2O                     45 ml
            sterile by autoclave
            add 1% RNase                  0.5ml
            store at room temperature

        Solution Ⅱ
        Cell lysis solution (0.2 N NaOH, 1% SDS)
            Mix 0.4 N NaOH and 2% SDS in same volume.

        Solution Ⅲ
        Neutralization solution (1.32 M potassium acetate, pH 5.2)
            5 M potassium acetate             13.2 ml
            ddH2O                       27 ml
            adjust pH to 5.2
            add ddH2O to                   50 ml
            sterile by autoclave
            store at room temperature



        三 DNA 操作试剂 - 返回 -
        1.5 × CTAB
           CTAB               15 g
           1 M Tris ・ Cl (pH 8.0)     75 ml
           0.5 M EDTA             30 ml
           NaCl                 61.4 g
           add ddH2O to             1000 ml

        0.5 M EDTA ( pH 8.0)
           EDTA-Na・2H2O           186.1 g
           NaOH                20 g
           Adjust to pH 8.0
           ddH2O to              1000 ml
           sterilize by autoclaving

        1 M Tris・HCl
                       pH 7.4     pH 7.6     pH 8.0
           Tris base        121.1 g     121.1 g    121.1 g
           Concentracted HCl    70 ml      64 ml      42 ml
           ddH2O to        1000 ml     1000 ml    1000 ml
           Sterilize by autoclaving

        TE ( pH 8.0)
                                    Stock          vol.
            10 mM Tris・HCl ( pH 8.0)       1 M            10 ml
            1 mM EDTA ( pH 8.0)           0.5 M          2 ml
            ddH2O to                              1000 ml
            sterilize by autoclaving

        10 M NH 4 Ac
           NH4Ac     385 g      770 g
           H2O to      500 ml    1000 ml

        10 × PCR buffer
                              stock             vol.
            500 mM KCl         2.5 M(sterilized)       200 ml
            100 mM Tris-HCl      1 M pH 9.0 (sterilized)     100 ml
            1% Triton X-100      100%               10 ml
            ddH2O                             690 ml
            sterilize by autoclaving

        5 × TBE
            Tris            54 g
            Boric acid          27.5 g
            0.5 M EDTA ( pH 7.9)   20 ml
            ddH2O to           1000 ml

        10 × TAE
            Tris              121.1 g       484.4 g
            EDTA(0.5 M)         20 ml        80 ml
            NaAc・3H2O          17 g        68 g
            glacial acetic acid    30 ml         200 ml
            adjust to pH 8.1
            ddH2O to           1000 ml       4000 ml

        NaOH
                         10 N         4 N
            NaOH           400 g         160 g
            ddH2O to        1000 ml        1000 ml

        2 N HCl
            concentrated HCl    365 ml     182.5 ml
            ddH2O to         2000 ml     1000 ml

        5 mg/ml ssDNA
           Salmon sperm DNA     1 g
           ddH2O to           200 ml

        0.5 M P.B (phosphate Buffer) pH 6.8
          Na2HPO4     16.44 g     131.52 g
          NaH2PO4     16.11 g    128.88 g
          ddH2O to    500 ml     4000 ml

        20 × SSC
           NaCl            175.3 g         701.2 g
           Na3Citrate        88.2 g          352.8 g
           ddH2O to         1000 ml          4000 ml
           Sterilize by autoclaving

        10% SDS
           SDS       100 g
           ddH2O to    1000 ml
           Heat to 68 ℃ to assist dissolution

        50 × Denhart's Solution
           Ficoll 400       10 g
           PVP-360         10 g
           BSA (Fraction V)    10 g
           ddH2O to 1000 ml

        Southern Blot Hybridization Buffer (Saghai , s Lab)
           Final conc.       Stock       Vol.
           5 × SSC          20 ×       250 ml
           50 mM PB (pH 6.8)      0.5 M         100 ml
           5 × Denhardt's       50 ×         100 ml
           2.5 mM EDTA (pH 8.0)       0.5 M               5 ml
           100 μg/ml ssDNA           5 mg/ml             20 ml
           0.4%SDS                    20%                 20 ml
           Dextran sulfate                                50 g
           ddH2O to                                       1000 ml
           (Place a beaker on a stirrer, add these solution in the order of appearance one by one. SDS should be the very last item.)

        Washing off Probe for Re-hybridization of Blots (I)
           Washing time: 10 min
           Final conc.       Stock       Vol.
           0.1 × SSC        20 × SSC      20 ml
           0.1% SDS         10% SDS       40 ml
           ddH2O to                     4000 ml

        Washing off Probe for Re-hybridization of Blots (II)
           Washing time: 3 min
           Final conc.       Stock       Vol.
           0.1 N NaOH        10 N NaOH     40 ml
           0.2% SDS         10% SDS       80 ml
           ddH2O to                   4000 ml

        Washing off Probe for Re-hybridization of Blots( Ⅲ )
           Washing time: 20 min
           Final conc.        Stock        Vol.
           0.2 M Tris. ( pH 7.5)   1 M Tris. (pH 7.5)   800 ml
           0.1 × SSC         20 × SSC       20 ml
           0.2% SDS          10% SDS        80 ml
           ddH2O to                     4000 ml
           Blue Juice

        Final conc.        Stock        Vol.       Vol.
           70% Glycerol       100%         35 ml       70 ml
           0.5 × TBE         5 ×        5 ml        10 ml
           0.2% SDS           10%          1 ml        2 ml
           20 mM EDTA         0.5 M        2 ml        4 ml
           5 mg/ml Bromphenol Blue             0.25 g       0.5 g
           5 mg/ml Xylene cyanol               0.25 g       0.5 g
           ddH2O to                     50 ml        100 ml

        EB (10 mg/ml)
           ehidium bromide        1 g
           ddH2O to            100 ml
           Stir on a magnetic stirrer for several hours. Transfer the solution to
           a dark bottle and store at 4 ℃ .
           The concentration of work solution: 0.5 μg/μl (50 μl stock solution
           In 1000 ml dH2O).
           Decontamination of EB
           Reduce the concentration of EB < 0.5 mg/ml, add 1 volume of
           0.5 M KMnO4 ,mix carefully then add 1 volume of 2.5 N HCl,
           mix carefully and allow the solution to stand at room temperature
           for several hours. Add 1 volume of 2.5 N NaOH, mix and discard



        四 RNA 操作试剂 - 返回 -
        ・Stock Solution:
             1M NaAc(PH7.0) : 82g NaAc 先加一定量 ddH 2 O ,用 NaOH 调 PH 值至 7.0 ,再用 ddH2O 定容至 1L, 灭菌
             0.5M EDTA(PH8.0):186.1g EDTA 先加一定量 ddH 2 O,用 NaOH 调 PH 值至 8.0, 再用 ddH2O 定容至 1L,灭菌
             10×MOPS buffer :( 用 DEPC 水配,再灭菌 )
             MOPS              41.85g
             1M NaAc(PH7.0)         50ml
             0.5M EDTA(PH8.0)        20ml
             先加一定量 DEPC 水 ,再用 4N NaOH 调 PH 至 7.0( 约加 7ml),再用 DEPC 水定容至 1L,灭菌

            1M NaH 2 PO 4 buffer ( PH7.2) :
             NaH2PO4                71g
             H3PO4(85%)           4ml
             用灭菌的 DEPC 水定容至 1L

            20×SSC:
             NaCl                 175.3g
             Na3Citrate             88.2g
             用 ddH2O 定容至 1L, 灭菌

            10%SDS:
             SDS                  100.0g
             用灭菌的 ddH2O 定容至 1L( 将水加热到 68℃ 有助于溶解 )

        ・Work Solution

           Sample buffer :
            Deionized formamide           1000ul
            10×MOPS buffer              200ul
            37% formaldehyde              320ul

           Blue juice (loading buffer):
            Glycerol                   70ml
            5×TBE                    10ml
            10%SDS                     2ml
            0.5M EDTA(PH8.0)               4ml
            Bromphenol Blue              0.5g
            Xylene Cyanol               0.5g
            加灭菌的 ddH2O 定容至 100ml

           Hybridization buffer:
            1M NaH2PO4buffer             500ml
            0.5M EDTA(PH8.0)              2ml
            10%SDS                     70g
            BSA                       10g
            用灭菌的 DEPC 水定容至 1L,贮存于室温下

           洗膜液 Ⅰ:( for 1 littre )
           20×SSC                   100ml
           10%SDS                     10ml

           洗膜液 Ⅱ : ( for 1 littre )
           20×SSC                   25ml
           10%SDS                    10ml

           洗膜液 Ⅲ:( for 1 littre )
           20×SSC                    5ml
           10%SDS                    10ml

           4×SSC :
           20×SSC                  200ml
           用灭菌的 DEPC 水定容至 1L

          2×SSC:
          20×SSC                  100ml
          用灭菌的 ddH2O 定容至 1L

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