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常用试剂

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1082

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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