分子生物学实验基本数据
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Units
-
1 mg = 10-3 g
1 ug = 10-6 g
1 ng = 10-9 g
1 pg = 10-12 g
-
1 kb of double stranded DNA = 660 kD
1 kb of single stranded DNA = 330 kD
1 kb of single stranded RNA = 340 kD
-
Average MW of a deoxynucleotide base = 324.5 Daltons
Average MW of a deoxynucleotide base pair = 649 Daltons
-
1 ug/ml of DNA = 3.08 uM phosphate
1 ug/ml of 1 kb of DNA = 3.08 nM 5' ends
1 mol of pBR322 = 2.83 x10+6 g.
1 pmol of linear pBR322, 5' ends = 1.4 ug
1 A260 unit of duplex DNA = 50 ug
1 A260 unit of single-stranded DNA = 37 ug
1 A260 unit of single-stranded RNA = 50 ug
-
1 kb of DNA = 333 amino acids of coding capacity
= 37,000 daltons
- Densities (50% GC):
RF I (supercoiled) ds DNA 1.709 g/ml RF II (nicked) ds DNA 1.54 g/ml ss DNA 1.726 g/ml ss RNA 1.90 g/ml protein 1.33 g/ml
Formulas
- DNA melting point :
-
For duplex DNA >50 bp:
Tm = 81.5deg. C +16.6 log (M of NaCl) + 0.41(% GC)
- [500/bp of shortest chain in duplex]
- [0.65(% formamide)]
For duplex DNA <50 bp:
Add 2deg. C for each A or T
Add 4deg. C for each G or C
- Picomoles of ends :
- pmol ends per ug linear DNA = 3030/number of bases
DNA mobility in gels
- Migration of marker dyes in native polyacrylamide non-denaturing gels
Gel % | Bromophenol blue (BP) | Xylene cyanole (XC) |
3.5 | 100 | 460 |
5.0 | 65 | 260 |
8.0 | 45 | 160 |
12.0 | 20 | 70 |
20.0 | 12 | 45 |
- Migration of marker dyes in polyacrylamide denaturing gels
Gel % | Bromophenol blue (BP) | Xylene cyanole (XC) |
5.0 | 35 | 130 |
6.0 | 26 | 106 |
8.0 | 19 | 75 |
10.0 | 12 | 55 |
20.0 | 8 | 28 |
- Relative positions of different DNA forms on Tris-acetate agarose gels
The exact distance between bands is influenced by percentage of agarose, time of electrophoresis, concentration of Ethidium bromide, degree of supercoiling and the size and complexity of the DNA.
Amino acid symbols
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or Aspartic acid Asx B Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glutamine or Glutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
Commonly used restriction enzymes and assay buffers
Enzyme | Isoschizomers | Buffer | Temp | Recognition Site | Cloning sites |
Aat II | med | 37 | GACGT/C | Aat II | |
Acc I | med | 37 | GT/(AC)(GT)AC | Acc I, Cla I | |
Aha III | Dra I | med | 37 | TTT/AAA | blunt |
Alu I | med | 37 | AG/CT | blunt | |
Asu II | 37 | TT/CGAA | Acc I, Cla I | ||
Ava I | med | 37 | C/YCGRG | Sal I, Xho I, Xma I | |
Ava II | med | 37 | G/G(AT)CC | ||
Bal I | low | 37 | TGG/CCA | blunt | |
BamH1 | med | 37 | G/GATCC | BamH1, Bgl II | |
Bgl I | med | 37 | GCCN4/NGGC | ||
Bgl II | low | 37 | A/GATCT | BamH1, Bgl II | |
BstE II | high | 60 | G/GTNACC | ||
BstN I | low | 55 | CC/(AT)GG | ||
Cla I | low | 37 | AT/CGAT | Acc I, Cla I | |
Dra I | Aha III | low | 37 | TTT/AAA | blunt |
EcoR1 | high | 37 | G/AATTC | EcoR1 | |
EcoRundefined | low | 37 | /AATT | EcoR1 | |
EcoRV | med | 37 | GAT/ATC | blunt | |
Hae I | low | 37 | (AT)GG/CC(TA) | blunt | |
Hae II | low | 37 | RGCGC/Y | ||
Hae III | med | 37 | GG/CC | blunt | |
Hha I | Cfo I, HinP1 | med | 37 | GCG/C | Hha I |
Hinc II | med | 37 | GTY/RAC blunt | ||
Hind III | med | 37-55 | A/AGCTT | Hind III | |
Hinf I | med | 37 | G/ANTC | ||
HinP1 | Cfo I, Hha I | low | 37 | G/CGC | Acc I, Cla I |
Hpa I | low | 37 | GTT/AAC | blunt | |
Hpa II | Msp I | low | 37 | C/CGG | Acc I, Cla I |
Kpn I | low | 37 | GGTAC/C | Kpn I | |
Mbo I | Sau3A | med | 37 | /GATC | BamH1, Bgl II |
Msp I | med | 37 | C/CGG | Acc I, Cla I | |
Mst I | Fsp I | high | 37 | TGC/GCA | blunt |
Mst II | Bsu36 I | high | 37 | CC/TNAGG | |
Nae I | med | 37 | GCC/CCG | blunt | |
Nco I | high | 37 | C/CATGG | Nco I | |
Nde I | med | 37 | CA/TATG | Nde I | |
Not I | high | 37 | GC/GGCCGC | ||
Nru I | med | 37 | TCG/CGA | blunt | |
Pst I | med | 21-37 | CTGCA/G | Pst I | |
Pvu I | high | 37 | CGAT/CG | Pvu I | |
Pvu II | med | 37 | CAG/CTG | blunt | |
Rsa I | med | 37 | GT/AC | blunt | |
Sac I | Sst I | low | 37 | GAGCT/C | Sac I, Sst I |
Sal I | high | 37 | G/TCGAC | Ava I, Sal I, Xho I | |
Sau3A I | Mbo I | med | 37 | /undefinedATC | BamH1, Bgl II |
Sfi I | 50 | GGCCN4/NGGCC | |||
Sma I | Xma I | (1) | 37 | CCC/GGG | blunt |
Sph I | high | 37 | GCATG/C | Sph I | |
Sst I | Sac I | med | 37 | GAGCT/C | Sst I, Sac I |
Sst II | Sac II | med | 37 | CCGC/GG | Sst II |
Taq I | low | 37-55 | T/CGA | AccI, Cla I | |
Tha I | FnuD II, Acc II | low | 37-60 | CG/CG | blunt |
Xba I | high | 37 | T/CTAGA | Xba I | |
Xho I | Ccr I | high | 37 | C/TCGAG | Ava I, Cla I |
Xma I | Sma I | low | 37 | C/CCGGG | Ava I, Xma I |
Assay buffers (see enzyme vendors catalogs for additional information)
10x Low salt buffer 10x Core buffer 100mM Tris-HCl, pH 7.6 500mM NaCl 100mM MgCl2 500mM Tris-HCl, pH 7.6 10mM DTT 100mM MgCl2 10x Medium salt buffer 10x Hind buffer 500mM NaCl 600mM NaCl 100mM Tris-HCl, pH 7.6 100mM Tris-HCl, pH 7.6 100mM MgCl2 70mM MgCl2 10mM DTT 10x High salt buffer 10x Sma I buffer (1) 1.0M NaCl 200mM KCl 500mM Tris-HCl, pH 7.6 100mM Tris-HCl, pH 7.6 100mM MgCl2 100mM MgCl2 10mM DTT 10mM DTT
Heat inactivation of restriction enzymes
- The following enzymes CAN be heat inactivated by incubation at 65 deg. C for 10 min.: Alu I, Apa I, Ava II, Bal I, Bgl I, Cvn I, Dpn I, Dra I, Eco R II, Eco RV, Hae II, Hha I, Hinc II, Kpn I, Mbo I, Msp I, Nar I, Nde II, Rsa I, Sau 3a, Sca I, Sfi I, Spe I, Sph I, Ssp I, Sst I, Stu I, and Sty I.
- The following enzymes are ONLY PARTIALLY heat inactivated by incubation at 65 deg.C for 10 min.: Ava I, Cfo I, Cla I, Cvn I, Eco RI, Mbo II, Mlu I, Nci I, Nru I, Pst I, Pvu II, Sma I and Xma III
- The following enzymes CANNOT be heat inactivated by incubation at 65 deg. C for 10 min.: Acc I, Bam HI, Bcl I, Bgl II, BstE II, Dde I, Hae III, Hind III, Hinf I, Hpa I, Hpa II Nde I, Nhe I, Nsi I, Pvu I, Sal I, Sau 96 I, Sst II, Taq I, Tha I, Xba I, Xho I, and Xor II.
Oligonucleotide universal primers used for DNA sequencing
M13 (-21) universal forward 5'-TGT-AAA-ACG-ACG-GCC-AGT-3'
M13 (-40) universal forward 5'-GTT-TTC-CCA-GTC-ACG-AC-3'
M13/pUC reverse primer 5'-CAG-GAA-ACA-GCT-ATG-ACC-3'
T7 primer 5'-TAA-TAC-GAC-TCA-CTA-TAG-GG-3'
SP6 primer 5'-ATT-TAG-GTG-ACA-CTA-TAG-3'
-16bs 5'-TCG-AGG-TCG-ACG-GTA-TCG-3'
+19bs 5'-GCC-GCT-CTA-GAA-CTA-GTG-3'
Listing of M13 (pUC) cloning sites
As they are read on DNA sequencing gels using the Universal primer:
M13mp7 .......EcoR1....BamH1.SalI..PstI..SalI..BamH1....EcoR1 GGCCAGTGAATTCCCCGGATCCGTCGACCTGCAGGTCGACGGATCCGGGGAATTC M13mp8 ..........HindIII.PstI.SalI...BamH1.SmaI.EcoR GGCCAGTGCCAAGCTTGGCTGCAGGTCGACGGATCCCCGGGAATTCGTAATCATG M13mp9 .......EcoR1.SmaI.BamH1..SalI..PstI..HindIII GGCCAGTGAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTTGGCGTAATCATG M13mp10 ...HindIII..PstI..SalI..XbaI..BamH1..SmaI..SstI..EcoR1 GCCAAGCTTGGGCTGCAGGTCGACTCTAGAGGATCCCCGGGCGAGCTCGAATTCG M13mp11 ...EcoR1..SstI..SmaI..BamH1..XbaI..SalI..PstI..HindIII GTGAATTCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGCCCAAGCTTGG M13mp18 HindIII.SphI..PstI..SalI.XbaI.BamH1.SmaI.KpnI.SstI.EcoR1 AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC M13mp19 EcoR1.SstI.KpnI.SmaI.BamH1.XbaI.SalI.PstI..SphI..HindIII GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTT