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分子生物学实验基本数据
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
EcoR1* 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 /G*ATC 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 |
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发表于 2006-9-29 11:00:50