分子微生物学方法总结
试验方法汇总 (DNA提取、纯化,克隆,T-RFLP酶切、乙醇沉淀、上机,质粒提取,RNA提取,PCR) 土壤DNA 提取方法:
试剂:——
TNS [(0.5M Tris-HCl; pH8.0; 0.1M NaCl; 10%SDS; 过滤除菌)
PB (120 mM, pH=8.0(300ml ) 112.87g Na2HPO4,7.12mM NaH2PO4; 高温灭菌) TE buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA; 高温灭菌)
PCI (苯酚:氯仿:异戊醇=25:24:1)
CI (氯仿:异戊醇=24:1)
异丙醇
70% 乙醇
器材:——
2 ml beat-beating tube
2 ml离心管
1.5 ml离心管
100 ul抢头
步骤——
0.5 g soil
2 ml beadsbeating tube
660 ul PB + 220 ul TNS
Fastprep 6.5m/s for 45s
14000 rpm,4℃,4 min
取750 ul上清液到2 ml离心管
加入750 ul PCI,手摇
14000 rpm,4℃,4 min
取600 ul上清液到新2ml 离心管
加入600 ul CI,手摇
14000 rpm,4℃,4 min
取上清液500 ul到新2 ml离心管
750异戊醇(-20℃)
-20℃过夜或至少保存2小时
14000 rpm,4℃,4 min
去上清液
500ul -20℃预冷70%乙醇,votex
14000 rpm,4℃,4 min
去上清液,挥发干乙醇
加入50-100 ul超纯水溶解DNA,-20℃待用。
DNA 纯化方法——
水饱和Sph-dex 40 (Sph dex 40:H2O=1:1)
步骤:
在BioSpin 柱加入1ml 水饱和Sph-dex 40
2000 rpm,4℃,1 min
重复一次(即:在BioSpin 柱加入1ml 水饱和Sph-dex 40
2000 rpm,4℃,1 min)
加入200-500 ul TE缓冲液
2000 rpm,4℃,1 min)
加入200-500ul DNA
2000 rpm,2min
所收集的即为DNA,-20℃保存待用。
克隆验证PCR——
H2O 19.5ul
Buffer (10*) 2.5ul
dNTP(10mM) 0.5 ul
M13-47(10uM)0.5ul
RVM (10uM)0.5ul
Tag enzyme 0.5ul
质粒DNA 1ul
Themocycling:
94℃ 3min
94℃ 60s
55℃ 60s
72℃ 90s
20-25cycles
72℃ 8min
克隆:——
试剂配制:——
IPTG 0.1 mol/L
LB
培养基和平板制作:——
胰蛋白胨 5g
酵母提取物2.5g
NaCl 5g
琼脂7.5g 去离子水定容到 500ml,121℃灭菌20min 冷却到60℃以下,加1ml 50mg/ml的Amp 溶液(终浓度
插入片段和载体的比例计算:——
(载体量ng ×插入片段大小kb)/载体大小kb ×3/1 = 插入片段ng
于超净台操作——
1, 在0.5 ml离心管中加入1ul pMD19-T vector和4ul Inert DNA
2, 加入5 ul(与上等量)Solution I
3, 16℃反应3-16 h
4, (同时准备振荡器和水浴锅)
5, 制作含Amp 的LB 平板,涂40 ul X-Gal和100ul IPTG
6, 提前10 min 拿出-80℃感受态细胞,冰浴溶化
7, 将1+2的反应液全部加入100ul 的JM109感受态细胞中,冰浴30min 8, 打开水浴锅42℃,振荡器培养箱37℃
9, 42℃水浴90s,冰置1min
10,加入890ul SOC培养基(预热37℃)
11,37℃振荡培养1h
12,5000rpm离心5min,去500ul 上清液(此步视纯化后DNA 浓度而定),混匀
13,涂板(10,30,60ul/板),倒置培养37℃,18h,剩余菌液4℃保存
14,挑取白色菌落100个至含Amp 的LB 板,划线,37℃倒置培养18h
T-RFLP——步骤:
荧光标记产物PCR
PCR 产物且胶纯化(蓝罡切胶纯化,电话?)
酶切——按内切酶说明书操作,
酶切产物纯化——
PCR 产物
加入1/10体积NaAc(pH=5.3,4℃)
2倍体积-20℃ 100%乙醇
-20℃过夜
14000rpm,15min,4℃
去上清液
100ul,-20℃ 70%乙醇,
14000rpm,15min,4℃
取上清液
挥发
-20℃保存待用
上机准备——
H.D. 8.8ul
内参 Rox or Liz 0.2ul
酶切产物 1ul
95℃,3min,冰上冷却
RNA 提取——
0.5g soil to 2ml beatbeading tube with
0.7g beads
Add 700 ul precooled TPM buffer (4degre)
Shaking at max speed for 45s
13000g at 4degree for 4min
Supernatant Pellet
add 700ul phenol based lysis buff shaking at max speed for 45s 13000g at 4degree for 10min Supernatant
To new 2ml tube
Add 500ul water satured phenol, mixing
13000g at 4degree for 4min
Supernatant
Add 500ul PCI, mixing
13000g at 4degree for 4min
Supernatant
Add 500ul CI
13000g at 4degree for 4min
Supernatant
3V EtOH or 1V 异丙醇and 0.01V Sodium acetate
-20degree,overnight or -80degree for 2h
13000g at 4degree for 1h
Remove EtOH
500ul EtOH 70%
13000g at 4degree for 10min
Remove 70%EtOH
Drying in air
Add DEPC water
Check with gel
DNAase Digestion
RNA sample
Add 1V TMC buffer,1ul RNAase free DNAase, 1ul RNAsin (RNA酶抑制剂,40U/ul) Incubate at 37degree for 45min
Add 1V CI
13000g at 4degree for 10min
Supernatant
3V EtOH or 1V异丙醇,0.1V Sodium acetate,mixing
Stored at -80degree for 1h
13000g at 4degree for 1h
Remove etOH
500ul 70%EtOH
13000g at 4degree for 10min
Air dried
200ul DEPC water
Check with gel
1. 500mM Tris-HCl (40ml): MW 157.6g/mol
m=500mM *40ml*157.6g/mol=3.152g (DEPC water)
2. 200mM Tris-HCl (50ml) pH7.5 autoclave (20ml for TPM buffer and 20ml for phenol-based lysis buffer)
20ml 500mM Tris-HCl + 30 ml DEPC water
3. TPM buffer (80ml) autoclave
50mM Tri-HCl: 20ml 200mM Tris-HCl
1.7%(w/v) polyvinylpyrrolidone K25: m=80*1.7*1/100=1.36g
PVP 聚乙烯吡咯烷酮
10mM MgCl2 =10*80*10^-6*203.31(MgCl2.6H2O)=0.163g
4. Phenol –based lysis buffer (80ml)
50mM Tris-HCL: 20ml 200mM Tris-HCL
10mM Na2EDTA: m=10*80*10^-6*372.24 =0.298
1% SDS
6% water satured phenol
5. sodium acetate (3M, PH5.2)
DEPC 处理水的方法:
1, add 0.1ml DEPC to 100ml of the solution to be treated and shake vigorously to
bring the DEPC into solution;
2, Let the solution incubate fro 12h at 37 degree;
3, Autoclave for 15min to remove any trace of DEPC;
注意:先把DEPC 配成水,再用DEPC 水配溶液。