常用试剂
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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
