SOP
6. 附录
Release time:2019-11-11 Browse:166

6 附录... 289

6.1.1常用抗生素配方... 289

6.1.2常用母液配方... 290

6.1.3缓冲液(pH 6-10)pKa及缓冲范围... 293

6.1.4磷酸缓冲液配方... 294

6.2.1常用网站... 296

6.2.2通用测序引物... 297

6.2.3密码子及其偏好性表格... 298

6.2.4空间群表格... 301

6.2.5线站新衍射仪BL17U1. 302

6.2.6实验室载体... 302

6.2.7实验室细胞株及菌株... 307

6.2.8结晶试剂盒.... 310

抗生素

母液浓度(mg/mL

工作浓度μg/mLfor

严紧型质粒

工作浓度(μg/mLfor

松驰型质粒

处理方式

Ampicillin,氨苄青霉素(Amp)

100(溶于水)

20

60

过滤除菌

Kanamycin,卡那霉素(Kan)

50 (溶于水)

10

50

过滤除菌/高压

Chloramphenicol,氯霉素(Chl/Cm/CAP)

34 (溶于乙醇)

 

25

170

无需灭菌

Carbenicillin,羧苄青霉素(Cab)

50 (溶于水)

20

60

过滤除菌

Streptomycin,链霉素(Sm)

10 (溶于水)

10

50

过滤除菌

Tetracyyline,四环素(Tet)

5 (溶于甲醇)

10

50

无需灭菌





















1.   保存条件:建议 -20 °C保存。

2.    mycin,霉素,在不严格的缩写中作为“XX霉素”的后缀缩写为m

3.    氯霉素(Chlor Am Phenicol),氯氨苯醇,标准缩写CAP,有时也写做Cm

4.    过滤灭菌:用0.22 μm一次性滤膜过滤除菌,于超净台内。

5.   母液配制方法举例,如100 mg/mL氨苄青霉素:溶解1 g氨苄青霉素钠盐于足量的水中,最后定容至10 mL。分装成小份于-20 °C贮存。常以25 μg/mL50 μg/mL的终浓度添加于生长培养基

1.   50× TAE1 L):

Tris                                                          242 g

Glacial Acetic Acid (冰醋酸)                 57.1 mL

0.5 M EDTA                                           100 mL

2.    10× TBE1 L):

Tris                                                           108.8 g       

Boric Acid                                                55.0 g             

0.5 M EDTA                                            40 mL                

3.    6×DNA Loading Buffer100 mL):

1 M Tris-HCl pH 7.4                                  10 mL

100% Glycerol                                           60 mL

0.5 M EDTA                                              12 mL

Bromophenol Blue                                     0.01 g

4.   0.5 M EDTA pH 8.0500 mL):

EDTA Disodium Salt Dihydrate               93.05 g  

Dissolve with about 700 mL ddH2O, stir, then adjust pH to 8.0 (1 M NaOH)

Adjust the volume to 1 L. Filtered with 0.22 μm membrane or autoclave.

**EDTA does not dissolve until nearly pH 8.0

5.   X-Gal 40 mg/mL, 1000×5 mL

X-Gal                                                          0.200 g

Dimethyl Foramide                                      5 mL

use glass pipette!

Make ~500 μL aliquots.  Store in brown glass vials at -20 °C.

6.   SOC medium250 mL

Tryptone Peptone                                          5 g

Yeast extract                                                  1.25 g

NaCl                                                              0.15 g

KCl10 mM                                            0.05 g

MgCl210 mM                                       0.51 g

50% glucose                                                   4 mL

add sterile and MgCl2 and glucose after autoclaving

7.    Solutions for Plasmid Preparations

1)    Qiagen Buffer P150 mM Tris-HCl pH 8.010 mM EDTA100 μg/mL RNaseA

&for resuspension of cells in DNA plasmid preparation. 和生工P1通用。

1 M Tris pH 8.0                                               5 mL       

0.5 M EDTA pH 8.0                                        2 mL

add ddH2O to 100 mL, Autoclave. Rnase A (add after autoclaving)  10.0 mg. Store at 4 °C

2)    Qiagen Buffer P2200 mM NaOH1% SDS

&for lysis of cells in DNA plasmid preparation. 和生工P2通用。

NaOH                                                               0.8 g

SDS                                                                  1.0 g

add ddH2O to 100 mL

3)     Qiagen Buffer P33.0 M potassium acetate pH 5.5,

&not for spin columns, but for Qiatips, midi, maxi, giga kits

Potassium Acetate                                  25.03 g

Acetic Acid, Galacial                ~11.5 mL

Adjust pH to 5.5, add ddH2O to 100 mL

4)     Qiagen Buffer N34.2 M Gu-HCl0.9 M potassium acetate pH 4.8

&as nutrealization buffer to precipitate proteins and genomic materials,和生工小抽试剂盒P3通用。

5)     Qiagen Buffer PB5 M Gu-HCl, 30% isopropanol)=PBII for pH indicator

6)     Qiagen Buffer QG5.5 M guanidine thiocyanate GuSCN),20 mM Tris HCl pH 6.6

7)     Qiagen Buffer PE10 mM Tris-HCl pH 7.580% ethanol

&for spin column salt wash in miniprep blueor pink columns. 用于质粒小抽和核酸提取离心柱清洗除盐。

8)     Qiagen Buffer QX17 M NaPO410 mM NaAc pH 5.3

&for solution and binding of agarose gels,用在用琼脂糖珠子DNA纯化试剂盒。

9)      Qiagen Buffer QXB5 M GuHCl

&for binding of large >3000 bp fragments to columns

10)     Qiagen Buffer QBT750 mM NaCl50 mM MOPS pH 7.015% isopropanol0.15% triton X-100

&Equilibration buffer

NaCl                                                                 4.38 g

Isopropanol                                                      15 mL

MOPS                                                               1.05 g

Triton X-100                                                     150 mL

Adjust pH to 7.0use 1N, add ddH2O to 100 mL

11)     Qiagen Buffer QC1.0 M NaCl50 mM MOPS pH 7.015% isopropanol

&Wash buffer

NaCl                                                                 5.84 g

MOPS                                                              1.05 g

Isopropanol                                                      15 mL

Adjust pH to 7.0, add ddH2O to 100 mL ,filter sterilize.

12)     Qiagen Buffer QF1.25 M NaCl 50 mM Tris-HCl pH 8.515% isopropanol

&Elution buffer

NaCl                                                            7.31 g

Tris 8.5 1 M                                                 5 mL

Isopropanol                                                 15 mL

Adjust pH to 8.5, add ddH2O to 100 mL

8.     Buffers for Protein Electrophoresis

1)    6×SDS Sample Buffer

1 M Tris-HCl pH 6.8                   25 mL

10% SDS                                     20 mL

50% Glycerol                              50 mL

1 M DTT                                      2 mL

Bromophenol Blue                     0.01 g

Add ddH2O to 100 mL

2)   Coommassie Blue Staining Solution500 mL):

Coommassie Brilliant Blue R250                     0.5 g

Ethanol                                                           200 mL

Acetic acid:                                                      50 mL

ddH2O                                                              to 500 mL~250 mL

Dissolve Coommassie in Ethanol before adding water and acetic acid will require stirring 1~2 hours

3)   Coommassie Blue Destaining Solution 500 mL):

Ethanol                                     200 mL                           

Acetic Acid                               50 mL

ddH2O                                      250 mL

4)    20×MES-SDS running buffer 1 L):MES2-(N-吗啉)乙磺酸一水合物

&For high-resolution separation of small size proteins.

MES                                                   195.2 g  

Tris                                                     121.2 g

SDS                                                      20 g

EDTA                                                     6 g

9.    Molecular Biology Buffers:

1)    A11×NEBuffer 1yellow):10 mM Bis Tris Propane-HCl10 mM MgCl21 mM DTT pH 7.0 @ 25 °CSupplied as a 10×concentrated stock.

2)    A21×NEBuffer 2blue):50 mM NaCl, 10 mM Tris-HCl10 mM MgCl21 mM DTT pH 7.9 @ 25 °CSupplied as a 10×concentrated stock.

3)   A21×NEBuffer 3red):100 mM NaCl, 50 mM Tris-HCl10 mM MgCl21 mM DTT pH 7.9 @ 25 °CSupplied as a 10×concentrated stock.

4)   A21×NEBuffer 4greencutsmart buffer):50 mM potassium acetate20 mM Tris acetate10 mM magnesium acetate1 mM DTT, pH 7.9 @ 25 °CSupplied as a 10×concentrated stock.

5)   2×T4 DNA quick ligase 快连接酶buffer60 mM Tris-HCl pH 7.820 mM MgCl22 mM DTT2 mM ATP and 10% PEG6000

6)   10×T4 DNA连接酶buffer500 mM Tris-HCl pH 7.6100 mM MgCl2100 mM DTT10 mM ATP

7)   10×pfu buffer200 mM Tris-HCl pH 8.6100 mM KCl160 mM (NH4)2SO410 mM MgCl21% Triton X-1001 mg/mL BSA

8)   5×HF buffer32 mM HEPES-KOH buffer pH 7.8100 mM KAcO, 4 mM Mg (AcO) 20.05% BSA0.5 mg/mL

9)   5×GC rich buffer32 mM HEPES-KOH buffer pH 7.8100 mM KAcO7.5 mM Mg(AcO)20.01% BSA0.1 mg/mL),1.0% DMSO

10)     2×Taq mix:

1 M Tris-HCl pH 8.6                               4 mL

1 M KCl                                                   20 mL

1 M MgCl2                                              600 μL

Sucrose                                                    68 g

dNTP 2 mM each                           40 mL

1% Cresol Red(甲酚红)                    2 mL

Taq polymerase 2U/μL                  20 mL

Add ddH2O to 200 mL

11)     10×Taq buffer:

200 mM Tris-HCl 8.4200 mM KC100 mM (NH4)2SO430 mM MgSO41 mg/mL BSA

6.1.3 缓冲液(pH 6-10)pKa及缓冲范围

缓冲液

pKa25°C

缓冲范围

分子量

中文全称

MES

6.1

5.5~6.7

195.2

2-吗啉乙磺酸

Bis-Tris

6.5

5.8~7.2

209.2

(2-羟乙基)亚胺基三(羟甲基)甲烷

ADA

6.6

6.0~7.2

190.2

N-(氨基甲酰基甲基)亚氨基二乙酸

ACES

6.8

6.1~7.5

182.2

N-氨基甲酰甲基乙磺酸

PIPES

6.8

6.1~7.5

302.4

哌嗪-NN'-(2-乙磺酸)

MOPSO

6.9

6.2~7.6

225.3

3-(N-吗啉基)-2-羟基丙磺酸

Bis-Tris Propane

6.8

6.3~9.5

282.3

[(羟甲基)氨基丙烷]/1,3-[(羟甲基)甲氨基]丙烷

BES

7.1

6.4~7.8

213.2

N-(2-羟乙基)-2-氨基乙璜酸

MOPS

7.2

6.5~7.9

209.3

3-(N-吗啡啉)乙磺酸

HEPES

7.5

6.8~8.2

238.3

N-2-羟乙基哌嗪-N'-2-乙磺酸

TES

7.4

6.8~8.2

229.2

N-3-(羟甲基)甲基-2-氨基乙磺酸

DIPSO

7.6

7.0~8.2

243.3

3-[N-N-(2-羟乙基)氨基]-2-羟基丙磺酸

TAPSO

7.6

7.0~8.2

259.3

N-3-(羟甲基)甲氨基-2-羟基丙烷磺酸

Tris

8.1

7.0~9.1

121.1

三羟甲基氨基甲烷

HEPPSO

7.8

7.1~8.5

268.3

4-(2-羟乙基)哌嗪-1-2-羟基丙磺酸

POPSO

7.8

7.2~8.5

362.4

哌嗪-1,4-二羟基丙磺酸

EPPS

8.0

7.3~8.7

252.3

4-羟乙基哌嗪丙磺酸

TEA

7.8

7.3~8.3

149.2

三乙醇胺

Tricine

8.1

7.4~8.8

179.2

N--(羟甲基)甲基氨基乙酸

Bicine

8.3

7.6~9.0

163.2

N,N-(2-羟乙基)甘氨酸

TAPS

8.4

7.7~9.1

243.3

三羟甲基甲胺基丙磺酸

AMPSO

9.0

8.3~9.7

227.3

3-[N-(11-二甲基-2-羟乙基)]氨基-2-羟丙烷磺酸

CHES

9.3

8.6~10.0

207.3

2-(环已胺)-1-乙磺酸

CAPSO

9.6

8.9~10.3

237.3

3-(环已胺)-2-羟基-1-丙磺酸

AMP

9.7

9.0~10.5

89.1

2-氨基-2-甲基-1-丙醇

CAPS

10.4

9.7~11.1

221.3

3-(环已胺)-1-丙磺酸

材料(MATERIALS

r  试剂(REAGENTS

NaH2PO4·2H2ONa2HPO4·12H2O

配制方法:

配制时,常先配制0.2 MNaH2PO40.2 MNa2HPO4,两者按一定比例混合即成0.2 M的磷酸盐缓冲液(PB),根据需要可配制不同浓度的PBPBS

1.  0.2 M NaH2PO4:称取NaH2PO4.2H2O 31.21 g(或 NaH2PO4·H2O 27.6 g)加重蒸水至1000 mL溶解。

2.  0.2 M Na2HPO4:称取Na2HPO4·12H2O 71.64 g(或 Na2HPO4·7H2O 53.6 g Na2HPO4·2H2O 35.61 g)加重蒸水至1000 mL溶解。

3.  0.2 M pH 7.4PB的配制:取19 mL 0.2 MNaH2PO481 mL 0.2 M Na2HPO4,充分混合即为0.2 MPBpH约为7.47.5)。若pH偏高或偏低,可通过改变二者的比例来加以调整,室温保存即可。

       25 °C0.2 M磷酸钠缓冲液的配制

pH

0.2 M Na2HPO4(mL)

0.2 M NaH2PO4(mL)

5.8

 8.0

 92.0

5.9

 10.0

 90.0

6.0

 12.3

 87.7

6.1

 15.0

 85.0

6.2

 18.5

 81.5

6.3

 22.5

 77.5

6.4

 26.5

 73.5

6.5

 31.5

 68.5

6.6

 37.5

 62.5

6.7

 43.5

 56.5

6.8

 49.0

 51.0

6.9

 55.0

 45.0

7.0

 61.0

 39.0

7.1

 67.0

 33.0

7.2

 72.0

 28.0

7.3

 77.0

 23.0

7.4

 81.0

 19.0

7.5

 84.0

 16.0

7.6

 87.0

 13.0

7.7

 89.5

 10.5

7.8

 91.5

 8.5

7.9

 93.5

 6.50

8.0

 94.7

 5.3

       25 °C0.1 M磷酸钾缓冲液的配制

pH

1 M K2HPO4(mL)

1 M KH2PO4(mL)

5.8

 8.5

 91.5

6.0

 13.2

 86.8

6.2

 19.2

 80.8

6.4

 27.8

 72.2

6.6

 38.1

 61.9

6.8

 49.7

 50.3

7.0

 61.5

 38.5

7.2

 71.7

 28.3

7.4

 80.2

 19.8

7.6

 86.6

 13.4

7.8

 90.8

 9.2

8.0

 94.0

 6.0

6.2 资源信息类

6.2.1 常用网站

常用网站根据功能分类如下:

1.         分子克隆(Molecular Cloning

1)         引物Tm值计算:http://biotools.nubic.northwestern.edu/OligoCalc.html

2)         密码子优化:https://sg.idtdna.com/CodonOpt

3)         质粒图谱分析:http://wishart.biology.ualberta.ca/PlasMapper/

4)         启动子信息:https://blog.addgene.org/plasmids-101-the-promoter-region

5)         addgene(一个质粒数据库):http://www.addgene.org/vector-database/

6)         NEB 内切酶buffer推荐:https://nebcloner.neb.com/#!/redigest

7)         核酸数据分析:http://biotools.nubic.northwestern.edu/OligoCalc.html

2.         蛋白表达与纯化(Protein Expression and Purification

1)         蛋白表达菌株信息:http://wolfson.huji.ac.il/expression/bac-strains-prot-exp.html

2)         蛋白纯化:http://wolfson.huji.ac.il/purification/Purification_Protocols.html

3.         蛋白序列分析(Protein Sequence Analysis

1)         信号肽预测:http://www.cbs.dtu.dk/services/SignalP/

2)         跨膜区预:TMpred http://www.ch.embnet.org/software/TMPRED_form.html

3)         结构域预测:http://smart.embl-heidelberg.de/

4)         蛋白性质预测:http://web.expasy.org/protparam/

              http://biotools.nubic.northwestern.edu/proteincalc.html

5)         蛋白质结构与功能预测:http://raptorx.uchicago.edu/

6)         二级结构预测:http://bioinf.cs.ucl.ac.uk/psipred/

7)         评估结构预测的准确性和可靠性:https://www.cameo3d.org/

8)         蛋白质结构和功能预测:https://open.predictprotein.org/

9)         三维结构比对http://ekhidna.biocenter.helsinki.fi/dali_server/start

10)     同源建模https://zhanglab.ccmb.med.umich.edu/I-TASSER/

11)     同源建模:https://swissmodel.expasy.org/

12)     预测蛋白相互作用:https://string-db.org/

13)     Pfam数据库是蛋白质家族的集合(根据结构域分类):http://pfam.xfam.org/

4.         结构分析Structure Analysis)

1)         RCSB PDB 数据库:http://www.rcsb.org/

2)         晶体界面分析 PISA serverhttp://www.ebi.ac.uk/pdbe/pisa/

3)         Deposition serverhttps://deposit-1.wwpdb.org/

4)         配体结构文件下载:http://www.rcsb.org/pdb/ligand/chemAdvSearch.do

5)         Bernhard Rupp的生物大分子晶体学网站http://www.ruppweb.org/Xray/101index.html

5.         其他

1)         ATCChttp://www.attc.org/

2)         化学分子性质:http://www.chemicalbook.com/ProductIndex_EN.aspx

3)         RNA修饰数据库:http://mods.rna.albany.edu/mods/modifications/

4)         Wikipediahttps://www.wikipedia.org/

5)         各类protocolshttp://openwetware.org/wiki/Protocols

6)         文献查询:https://sci-hub.tw/

6.2.2 通用测序引物

简介(INTRODUCTION

当进行菌落PCR时,如果目的片段没有成功转化至细菌中,针对插入片段的特异性引物可能会与细菌自身DNA进行非特异性结合,产生非特异性条带,从而对我们鉴定克隆是否成功构建产生干扰。因此我们常用载体上的引物进行鉴定。利用这种引物,我们既可以检测转入的质粒是否是空载体,也能估测连入载体的插入片段大小是否正确。

以下是我们实验室常用的几个通用引物,既可以做菌落PCR,也可以用于测序。

Primer name

Site

Sequence

Z5

T7 promoter

TACGACTCACTATAGGGGAATTGTGAGCG

Z6

T7 terminator

Z8

TEV and Sal I

gagaatctttatttccaaggttctgTCGAC

Z9

v52 Rev

GGCCAGTGAATTGTAATACGACTCACTATA

Z10

v55 Rev

GTTCTGATTTAATCTGTATCAGGCTGAAAA

Z11

pCold (v113, v114) Rev

aaatggcagggatcttagattctgtg

XY27

pcDNA3.1 For

taatacgactcactatagggagacccaagc

XY28

pcDNA3.1 Rev

tagaaggcacagtcgaggctgatca

y936

v108 Rev

ggctgattatgatcctctagtacttc

Z12

v104 For

aatttgtaatacgactcactatagggcga

Z13

v104 Rev

tttagaggccccaaggggttatgctagtt

Z14

v105 For

agatctttaatacgactcactatagggcga

Z15

v105 Rev

ttgagatggtgcacgatgcacagttgaa

Z16

V126, 127 For

agaacccactgcttactggcttatcgaaat

Z17

V126, 127 Rev

ctgatcagcgggtttaaactcaatggtgat

L22

pEGFP-N1 For

tctatataagcagagctggtttagtgaaccgtcag

L23

pEGFP-N1 Rev

tgcacgccgtaggtcagggtggtcac

L28

pCDH For

GAGCTCGTTTAGTGAACCGTCAGATCGCCT

L29

pCDH Rev

GTTCAATTGCCGACCCCTCCCCCCAACTTC

第一位碱基

第二位碱基

U

C

A

G

U

UUU (Phe/F)苯丙氨酸

UCU (Ser/S)丝氨酸

UAU (Tyr/Y)酪氨酸

UGU (Cys/C)半胱氨酸

UUC (Phe/F)苯丙氨酸

UCC (Ser/S)丝氨酸

UAC (Tyr/Y)酪氨酸

UGC (Cys/C)半胱氨酸

UUA (Leu/L)亮氨酸

UCA (Ser/S)丝氨酸

UAA (终止)

UGA (终止)

UUG (Leu/L)亮氨酸

UCG (Ser/S)丝氨酸

UAG (终止)

UGG (Trp/W)色氨酸






C

CUU (Leu/L)亮氨酸

CCU (Pro/P)脯氨酸

CAU (His/H)组氨酸

CGU (Arg/R)精氨酸

CUC (Leu/L)亮氨酸

CCC (Pro/P)脯氨酸

CAC (His/H)组氨酸

CGC (Arg/R)精氨酸

CUA (Leu/L)亮氨酸

CCA (Pro/P)脯氨酸

CAA (Gln/Q)谷氨酰胺

CGA (Arg/R)精氨酸

CUG (Leu/L)亮氨酸

CCG (Pro/P)脯氨酸

CAG (Gln/Q)谷氨酰胺

CGG (Arg/R)精氨酸






A

AUU (Ile/I)异亮氨酸

ACU (Thr/T)苏氨酸

AAU (Asn/N)天冬酰胺

AGU (Ser/S)丝氨酸

AUC (Ile/I)异亮氨酸

ACC (Thr/T)苏氨酸

AAC (Asn/N)天冬酰胺

AGC (Ser/S)丝氨酸

AUA (Ile/I)异亮氨酸

ACA (Thr/T)苏氨酸

AAA (Lys/K)赖氨酸

AGA (Arg/R)精氨酸

AUG (Met/M)甲硫氨酸

ACG (Thr/T)苏氨酸

AAG (Lys/K)赖氨酸

AGG (Arg/R)精氨酸






G

GUU (Val/V)缬氨酸

GCU (Ala/A)丙氨酸

GAU (Asp/D)天冬氨酸

GGU (Gly/G)甘氨酸

GUC (Val/V)缬氨酸

GCC (Ala/A)丙氨酸

GAC (Asp/D)天冬氨酸

GGC (Gly/G)甘氨酸

GUA (Val/V)缬氨酸

GCA (Ala/A)丙氨酸

GAA (Glu/E)谷氨酸

GGA (Gly/G)甘氨酸

GUG (Val/V)缬氨酸

GCG (Ala/A)丙氨酸

GAG (Glu/E)谷氨酸

GGG (Gly/G)甘氨酸


 

密码子偏好性表格:

 

人类密码子偏好性表格

第一位碱基

第二位碱基

第三位碱基

U

C

A

G

U

UUU17.6%

UCU15.2%

UAU12.2%

UGU10.6%

U

UUC20.3%

UCC (17.7%)

UAC15.3%

UGC (12.6%)

C

UUA7.7%

UCA12.2%

UAA1%

UGA (1.6%)

A

UUG12.9%

UCG4.4%

UAG (0.8%)

UGG (13.2%)

G

C

 

CUU (13.2%)

CCU17.5%

CAU10.9%

CGU4.5%

U

CUC 19.6%)

CCC19.8%

CAC15.1%

CGC10.4%

C

CUA (7.2%)

CCA16.9%

CAA12.3%

CGA6.2%

A

CUG39.6%

CCG6.9%

CAG34.2%

CGG11.4%

G

A

AUU16%

ACU (13.1%)

AAU17%

AGU12.1%

U

AUC (20.8%)

ACC18.9%

AAC19.1%

AGC19.5%

C

AUA7.5%)

ACA15.1%

AAA24.4%

AGA12.2%

A

AUG22%

ACG6.1%

AAG31.9%

AGG12%

G

G

GUU11%

GCU18.4%

GAU21.8%

GGU10.8%

U

GUC14.5%

GCC27.7%

GAC25.1%

GGC22.2%

C

GUA (7.1%)

GCA15.8%

GAA29%

GGA16.5%

A

GUG (28.1%)

GCG7.4%

GAG39.6%

GGG16.5%

G

 


 

大肠杆菌密码子偏好性表格

第一位碱基

第二位碱基

第三位碱基

U

C

A

G

U

UUU24.4%

UCU13.1%

UAU21.6%

UGU5.9%

U

UUC13.9%

UCC (9.7%)

UAC11.7%

UGC (5.5%)

C

UUA17.4%

UCA13.1%

UAA2%

UGA (1.1%)

A

UUG12.9%

UCG8.2%

UAG (0.3%)

UGG (13.4%)

G

C

 

CUU (14.5%)

CCU9.5%

CAU12.4%

CGU15.9%

U

CUC9.5%)

CCC6.2%

CAC7.3%

CGC14%

C

CUA (5.6%)

CCA9.1%

CAA14.4%

CGA4.8%

A

CUG37.4%

CCG14.5%

CAG26.7%

CGG7.9%

G

A

AUU29.6%

ACU (13.1%)

AAU29.3%

AGU13.2%

U

AUC (19.4%)

ACC18.9%

AAC20.3%

AGC14.3%

C

AUA13.3%)

ACA15.1%

AAA37.2%

AGA7.1%

A

AUG23.7%

ACG13.6%

AAG15.3%

AGG4%

G

G

GUU21.6%

GCU18.9%

GAU33.7%

GGU23.7%

U

GUC13.1%

GCC21.6%

GAC17.9%

GGC20.6%

C

GUA (13.1%)

GCA23%

GAA35.1%

GGA13.6%

A

GUG (19.9%)

GCG21.1%

GAG19.4%

GGG12.3%

G

 

6.2.4 空间群表格

 

6.2.5 线站新衍射仪BL17U1

简介(INTRODUCTION

上海光源BL17U1线站新衍射仪(自主研制,水平方向SOC~±0.342 μm,垂直方向SOC~±0.319 μm)和新探测器(瑞士Dectris公司的Eiger X 16 M)已经安装调试完成,1110号开始正式对用户开放使用。但是在开放初期还需要各位老师提醒实验人员注意以下事项:

1.         由于衍射仪测角头电机比较小,如果手动上样的话很容易把位置碰歪,所以目前只能机械手上样,建议各位老师将晶体样品保存在puck里,如果没有puck,可以提前到线站借用我们线站的puck将晶体样品转移到puck里再通过机械手上样。

2.         如果在实验的过程中发生故障,一定要第一时间给值班人员打电话,不能自行去处理。

3.          建议在EIGER 16 M探测器上收数据的策略是:0.1~0.3度一张,曝光时间0.05秒一张。这样收一套360度的完整数据(3600张衍射图)大概只需要3分钟。

4.         一套3600张衍射图的数据大小是25 GB左右,建议带一个存储空间大的硬盘拷贝数据,可以边实验边拷贝数据。减少拷贝数据等待时间。

5.         数据处理建议使用XDS

6.         目前线站的新系统还需要进一步优化调试,能量暂时固定在12.660 Kev

Name

Backnone

Description

Cloning Frame

Fusion Tag

Sequencing Primer 1

Sequencing Primer2

Resistance

  

  

V1

pET30a

NdeI-SalI,NotI-XhoI-His6-stop

SalI-NotI

none

T7 Promoter

T7 Terminator

Kan

  

  

V2

pET30a

NdeI-His8-SalI,NotI-stop

SalI-NotI

none

T7 Promoter

T7 Terminator

Kan

  

  

V3

pET30a

NdeI-His8-GB1-TEV-SalI,NotI-stop

SalI-NotI

His8-GB1-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V4

pET30a

NdeI-His8-GB1-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

His8-GB1-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V5

pET30a

NdeI-His8-TEV-SalI,NotI-stop

SalI-NotI

MGSS-His8-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V6

pET30a

NdeI-His8-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

MGSS-His8-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V7

pET30a

NdeI-His8-SUMO-TEV-SalI,NotI-XhoI-stop

SalI-NotI

MGSS-His8-SUMO-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V8

pET30a

NdeI-His8-VHb-TEV-SalI,NotI-stop

SalI-NotI

His8-VHb-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V9

pET30a

NdeI-His8-VHb-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

His8-VHb-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V10

pET30a

NdeI-His8-GST-TEV-SalI,NotI-stop

SalI-NotI

His8-GST-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V11

pET30a

NdeI-His8-GST-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

His8-GST-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V12

pET30a

NdeI-His7-TRX-TEV-SalI,NotI-stop

SalI-NotI

His7-TRX-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V13

pET30a

NdeI-His7-TRX-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

His7-TRX-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V14

pET30a

NdeI-His8-MBP-SalI,NotI-stop

SalI-NotI

His8-MBP

T7 Promoter

T7 Terminator

Kan

  

  

V15

pET30a

NdeI-His8-tag2-TEV-SalI,NotI-stop

SalI-NotI

His8-tag2-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V16

pET30a

NdeI-His8-tag2-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

His8-tag2-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V17

pET30a

NdeI-DsbA-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

DsbA-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V18

pET30a

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V19

pET30a

NdeI-His8-NusA-TEV-SalI,NotI-stop

SalI-NotI

His8-NusA-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V20

pET30a

NdeI-His8-GB1-TEV-SalI,NotI-Acidic   Peptide-LacZ-XhoI-His6-stop

SalI-NotI

His8-GB1-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V21

pET30a

NdeI-His8-MBP-helical linker-SalI,NotI-stop

SalI-NotI

His8-MBP-helical linker

T7 Promoter

T7 Terminator

Kan

  

  

V22

pMAL

NdeI-MBP-SalI,NotI-XhoI-LacZ-stop

SalI-NotI

MBP

M13R-pUC

M13F-pUC

Amp

  

  

V23

pMAL

NdeI-MBP-SalI,NotI-XhoI-LacZ-stop

SalI-NotI

MBP

M13R-pUC

M13F-pUC

Amp

  

  

V24

pMAL

NdeI-MBP-SalI,NotI-XhoI-LacZ-stop

SalI-NotI

MBP

M13R-pUC

M13F-pUC

Amp

  

  

V25

pMAL

NdeI-MBP-SalI,NotI-XhoI-LacZ-stop

SalI-NotI

MBP

M13R-pUC

M13F-pUC

Amp

  

  

V26

pMAL

NdeI-MBP-SalI,NotI-XhoI-LacZ-stop

SalI-NotI

MBP

M13R-pUC

M13F-pUC

Amp

  

  

V27

pMAL

NdeI-MBP-SalI,NotI-XhoI-LacZ-stop

SalI-NotI

MBP

M13R-pUC

M13F-pUC

Amp

  

  

V28

pET30a

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V29

pET30a

NdeI-MBP-TEV-SalI,NotI-stop

SalI-NotI

MBP-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V29H

pET30a

NdeI-His8-MBP-TEV-SalI,NotI-stop

SalI-NotI

His8-MBP-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V30

pET30a

NdeI-MBP-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V31

pLEXSY-Ble

NcoI-SP-SalI,NotI-His6-stop

SalI-NotI

Signal Peptide

P14

A26

Amp/Ble/Tet

  

  

V32

pLEXSY-Ble

NcoI-SP-Strep   Tag-His8-TEV-SalI,NotI-His6-stop

SalI-NotI

SP-Strep Tag-His8-TEV

P14

A26

Amp/Ble/Tet

  

  

V33

pLEXSY-Ble

NcoI-SalI,NotI-His8-stop

SalI-NotI

none

P14

A26

Amp/Ble/Tet

  

  

V34

pLEXSY-Ble

NcoI-Strep Tag-SalI,NotI-His8-stop

SalI-NotI

Strep Tag

P14

A26

Amp/Ble/Tet

  

  

V35

pLEXSY-Ble

NcoI-His8-GST-TEV-SalI,NotI-stop

SalI-NotI

His8-GST-TEV

P14

A26

Amp/Ble/Tet

  

  

V36

pLEXSY-Ble

NcoI-SP-Strep-His8-TEV-SalI,NotI-His6-stop

SalI-NotI

SP-Strep-His8-TEV

P14

A26

Amp/Ble/Tet

  

  

V37

pLEXSY-Ble

NcoI-Strep-His8-TEV-SalI,NotI-His6-stop

SalI-NotI

Strep-His8-TEV

P14

A26

Amp/Ble/Tet

  

  

V40

pTT5

NcoI-His8-GB1-TEV-SalI,NotI-stop

SalI-NotI

His8-GB1-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V41

pTT5

NcoI-His6-Strep-His8-TEV-SalI,NotI-stop

SalI-NotI

His6-Strep-His8-TEV

P14

A26

Amp/Ble/Tet

  

  

V42

pTT5

NcoI-Strep-His8-TEV-SalI,NotI-stop

SalI-NotI

Strep-His8-TEV

P14

A26

Amp/Ble/Tet

  

  

V51

pFab

EcoRV-SP-Strep-His8-TEV-SalI,NotI-His6-stop

SalI-NotI

SP-Strep-His8-TEV

unknown

unknown

Amp

  

  

V52

pFab

EcoRV-SP-His6-TEV-SalI,NotI-stop

SalI-NotI

SP-His6-TEV

unknown

unknown

Amp

  

  

V53

pFab

EcoRV-SP-His6-TEV-SalI,NotI-stop

SalI-NotI

SP-His6-TEV

unknown

unknown

Amp

  

  

V54

pFab

EcoRV-SP-His6-TEV-SalI,NotI-Strep-stop

SalI-NotI

SP-His6-TEV

unknown

unknown

Amp

  

  

V55

pMAL

NdeI-MBP-TEV-SalI,NotI-stop

SalI-NotI

MBP-TEV

M13R-pUC

M13F-pUC

Amp

  

  

V56

pMW103

LexA-SalI,NotI-stop

SalI-NotI

LexA

unknown

unknown

Kan

For Y2H

  

V57

pET30a

NdeI-hDim2-SalI,NotI-XhoI-His6-stop

SalI-NotI

hDim2

T7 Promoter

T7 Terminator

Kan

  

  

V59

pET30a

NdeI-His8-TEV-SalI,NotI-Lysozyme-stop

SalI-NotI

His8-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V60

pET30a

NdeI-Lysozyme-SalI,NotI-XhoI-His6-stop

SalI-NotI

Lysozyme

T7 Promoter

T7 Terminator

Kan

  

  

V61

pEU-01

His6-TEV-SalI,NotI-stop

SalI-NotI

His6-TEV

unknown

unknown

Amp

  

  

V62

pET30a

NdeI-MBP-SalI,NotI-lysozyme-XhoI-His6-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V63

pET30a

NdeI-MBP-TEV-SalI,NotI-Lysozyme-XhoI-His6-stop

SalI-NotI

MBP-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V64

pET30a

NdeI-His8-MBP-TEV-SalI,NotI-Lysozyme-stop

SalI-NotI

His8-MBP-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V65

pJG4-5

Gal1-B42-HA epitope-EcoRI-SalI,NotI-stop

SalI-NotI

Gal1-B42-HA epitope

unknown

unknown

Amp

For Y2H

  

V66

pET30a

NdeI-T4LS5-SalI,NotI-XhoI-His6-stop

SalI-NotI

T4LS5

T7 Promoter

T7 Terminator

Kan

  

  

V67

pEU-01

NdeI-His8-NcoI-TEV-SalI,NotI-stop

SalI-NotI

His8-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V71

pET30a

NdeI-SalI,NotI-XhoI-His6-stop

SalI-NotI

none

T7 Promoter

T7 Terminator

Kan

  

  

V72

pET30a

NdeI-SalI,NotI-Strep-stop

SalI-NotI

none

T7 Promoter

T7 Terminator

Kan

  

  

V77

pBAD

NdeI-SalI,NotI-XhoI-His6-stop

SalI-NotI

none

pBAD for

pBAD rev

Amp

  

  

V78

pBAD-V28E

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

pBAD for

pBAD rev

Amp

  

  

V79

pBAD-V29H

NdeI-His8-MBP-TEV-SalI,NotI-stop

SalI-NotI

His8-MBP-TEV

pBAD for

pBAD rev

Amp

  

  

V80

pCMV-HA

HAtag-EcoRI-SalI,NotI-stop

SalI-NotI

HAtag

pCMV

M13R-20

Amp

  

  

V81

pBAD-V30

NdeI-MBP-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP-TEV

pBAD for

pBAD rev

Amp

  

  

V82

pBAD-V29HS

NdeI-His8-MBP-TEV-SalI,NotI-strep-stop

SalI-NotI

His-MBP-TEV

pBAD for

pBAD rev

Amp

  

  

V83

pASK-17plus

NdeI-Strep-TEV-EcoRI-BamHI-XhoI-SalI-NcoI-EcoRV-HindIII

MCS

Strep-TEV

pASk for

pASK rev

Amp

  

  

V84

pASK-V3

NdeI-His8-GB1-TEV-SalI,NotI-stop

SalI-NotI

His8-GB1-TEV

pASk for

pASK rev

Amp

  

  

V85

pASK-V28E

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

pASk for

pASK rev

Amp

  

  

V86

pASK-V29H

NdeI-His8-MBP-TEV-SalI,NotI-stop

SalI-NotI

His-MBP-TEV

pASk for

pASK rev

Amp

  

  

V87

pASK-V30

NdeI-MBP-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP-TEV

pASk for

pASK rev

Amp

  

  

V88

pASK-V29HS

NdeI-His8-MBP-TEV-SalI,NotI-Strep-stop

SalI-NotI

His-MBP-TEV

pASk for

pASK rev

Amp

  

  

V89

pET30a

NdeI-InfB21-eGFP-SalI,NotI-stop

SalI-NotI

InfB21-eGFP

T7 Promoter

T7 Terminator

Kan

  

  

V90

pET30a

NdeI-InfB21-eGFP-TEV-SalI,NotI-stop

SalI-NotI

InfB21-eGFP

T7 Promoter

T7 Terminator

Kan

  

  

V91

pET30a

NdeI-His8-eGFP-TEV-SalI,NotI-stop

SalI-NotI

His8-eGFP-TEV

T7 Promoter

T7 Terminator

Kan

  

  

V92

pET30a

NdeI-SUMO-SalI,NotI-Strep-stop

SalI-NotI

SUMO

T7 Promoter

T7 Terminator

Kan

  

  

V93

pET30a

NdeI-His8-MBP-TEV-GFP-SalI,NotI-Strep-stop

SalI-NotI

His8-MBP-TEV-GFP

T7 Promoter

T7 Terminator

Kan

  

  

V94

pET30a

NdeI-VHb-SalI,NotI-Strep-stop

SalI-NotI

VHb

T7 Promoter

T7 Terminator

Kan

  

  

V95

pET30a

NdeI-Tag2-SalI,NotI-Strep-stop

SalI-NotI

Tag2

T7 Promoter

T7 Terminator

Kan

  

  

V96

pET30a

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V97

pET30a

NdeI-MBP-SalI,NotI-Strep-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V98

pFab

EcoRV-SP-NdeI-His8-SalI,NotI-Strep-stop

SalI-NotI

SP-His8

unknown

unknown

Amp

  

  

V99

pET30a

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V100

pET30a

NdeI-MBP-SalI,NotI-XhoI-His6-stop

SalI-NotI

MBP

T7 Promoter

T7 Terminator

Kan

  

  

V101

pET30a

NdeI-GlyA-SalI,NotI-XhoI-His6-stop

SalI-NotI

GlyA

T7 Promoter

T7 Terminator

Kan

  

  

V102

pET30a

NdeI-GacH-SalI,NotI-XhoI-His6-stop

SalI-NotI

GacH

T7 Promoter

T7 Terminator

Kan

  

  

V103

pET30a

NdeI-XBP1-SalI,NotI-XhoI-His6-stop

SalI-NotI

XBP1

T7 Promoter

T7 Terminator

Kan

  

  

V104

pGBKT7SN

DNA Binding-XhoI-NdeI-SalI,NotI-stop

SalI-NotI

DNA Binding

T7 Promoter

ADH

Kan

  

  

V105

pGADT7SN

SV40-AD-NcoI-Epitope-NdeI-SalI,NotI-stop

SalI-NotI

SV40-AD-Epitope

T7 Promoter

ADH

Amp

  

  

V106

pRmHa3

EcoRI-TEV-SalI,NotI-XhoI-His6-stop

SalI-NotI

TEV

pRmHa3 for

pRmHa3 rev

Amp

  

  

V107

pVL1392

NcoI-NotI-HIs6-stop

unknown

none

y326 polyhedrin

y327 polyhedrin

Amp

  

  

V108

plSUMOstar

HIs6-SUMOstar-SalI,NotI-stop

SalI-NotI

SUMOstar

unknown

unknown

Amp/Get

  

  

V109

pFastBacHT

NdeI-InfB21-His8-TEV-SalI,NotI-stop

SalI-NotI

InfB21-His8-TEV

pASk for

pASK rev

Amp/Get

  

  

V110

pFastBacHT

NdeI-SalI,NotI-XhoI-His6-stop

SalI-NotI

none

pASk for

pASK rev

Amp/Get

  

  

V111

pFastBacHT

NdeI-His8-MBP-TEV-Sali,NotI-stop

SalI-NotI

His8-MBP-TEV

pASk for

pASK rev

Amp/Get

  

  

V112

pFastBacHT

NdeI-His8-MBP-TEV-Sali,NotI-XhoI-His6-stop

SalI-NotI

His8-MBP-TEV

pASk for

pASK rev

Amp/Get

  

  

V113

pColdII

NdeI-His8-MBP-TEV-SalI,NotI-stop

SalI-NotI

His8-MBP-TEV

pCold for

pCold rev

Amp

  

  

V114

pColdII

NdeI-MBP-TEV-SalI,NotI-stop

SalI-NotI

MBP-TEV

pCold for

pCold rev

Amp

  

  

V115

pColdII

NdeI-XhoI-His6-stop

unknown

none

pCold for

pCold rev

Amp

  

  

V116

pET30a

NdeI-B562-XhoI-His6-stop

unknown

B562

T7 Promoter

T7 Terminator

Kan

  

  

V117

pCold

  

  

  

  

  

  

  

  

V118

pGADzaA

  

  

  

  

  

  

  

  

V119

FBQH

His8-TEV-BamHI-XhoI

BamHI-XhoI

His8-TEV

FBQ-F

FBQ-R

Amp

  

  

V120

FBQH

GST-TEV-BamHI-Xho I

BamHI-XhoI

GST-TEV

FBQ-F

FBQ-R

Amp

  

  

V121

FBQH

His-MBP-TEV-BamHI-XhoI

BamHI-XhoI

His-MBP-TEV

FBQ-F

FBQ-R

Amp

  

  

V122

PET30a

NdeI- His8- GB1 -Sal I -Not I-stop

Sal I-Not I

His8- GB1

T7-P

T7-T

Kan

XY

  

V123

pCDNA3.1

CMV-T7-FLAG-MCS-Stop

XhoI-BamH I-EcoR I-Not I

FLAG

T7/CMV

BGH-R

Amp

XY

  

V124

pCDNA3.1

CMV-T7-HA-MCS-Stop

XhoI-BamH I-EcoR I-Not I

HA

T7/CMV

BGH-R

Amp

XY

  

V125

pCDNA3.1

CMV-T7-Strep-MCS-Stop

XhoI-BamH I-EcoR I-Not I

Strep

T7/CMV

BGH-R

Amp

XY

  

V126

pCDNA3.1

CMV-T7-SP-3XFLAG-MCS-Stop

XhoI-BamH I-EcoR I-Not I

3XFLAG

T7/CMV

BGH-R

Amp

XY

SP=signal peptide

V127

pCDNA3.1

CMV-T7-SP-FLAG-Clover-MCS-Stop

XhoI-BamH I-EcoR I-Not I

Clover/FLAG

T7/CMV

BGH-R

Amp

XY

SP=signal peptide

V128

pCDNA3.1

CMV-T7-SP-HA-MCS-Stop

XhoI-BamH I-EcoR I-Not I

HA

T7/CMV

BGH-R

Amp

XY

SP=signal peptide

V129

pCDNA3.1

CMV-T7-SP-Strep-mRubby2-MCS-Stop

XhoI-BamH I-EcoR I-Not I

Strep/mRubby2

T7/CMV

BGH-R

Amp

XY

SP=signal peptide

V130

pCDNA3.1

CMV-T7-SP-MCS-Stop

XhoI-BamH I-EcoR I-Not I

Strep

T7/CMV

BGH-R

Amp

XY

  

V131

pCDNA3.1

CMV-T7-Strep-mRubby2-MCS-Stop

XhoI-BamH I-EcoR I-Not I

Strep/mRubby2

T7/CMV