分子生物学实验基本数据

• 1 mg = 10-3 g1 ug = 10-6 g1 ng = 10-9 g1 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 DaltonsAverage MW of a deoxynucleotide base pair = 649 Daltons

• 1 ug/ml of DNA = 3.08 uM phosphate1 ug/ml of 1 kb of DNA = 3.08 nM 5' ends1 mol of pBR322 = 2.83 x10+6 g.1 pmol of linear pBR322, 5' ends = 1.4 ug1 A260 unit of duplex DNA = 50 ug1 A260 unit of single-stranded DNA = 37 ug1 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

• 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 TAdd 4deg. C for each G or C
• Picomoles of ends:

• pmol ends per ug linear DNA = 3030/number of bases

• 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.

Alanine                         Ala     AArginine                        Arg     RAsparagine                      Asn     NAspartic acid           Asp     DAsparagine or Aspartic acid     Asx     BCysteine                        Cys     CGlutamine                       Gln     QGlutamic acid                   Glu     EGlutamine or Glutamic acid      Glx     ZGlycine                       Gly     GHistidine                       His     HIsoleucine                      Ile     ILeucine                 Leu     LLysine                  Lys     KMethionine                      Met     MPhenylalanine                   Phe     FProline                 Pro     PSerine                  Ser     SThreonineThr     TTryptophan                      Trp     WTyrosine                        Tyr     YValine                  Val     V

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 buffer10x Core buffer   100mM Tris-HCl, pH 7.6500mM NaCl   100mM MgCl2500mM Tris-HCl, pH 7.6    10mM DTT100mM MgCl210x Medium salt buffer10x Hind buffer   500mM NaCl600mM NaCl   100mM Tris-HCl, pH 7.6100mM Tris-HCl, pH 7.6   100mM MgCl2 70mM MgCl2    10mM DTT10x High salt buffer10x Sma I buffer (1)    1.0M NaCl200mM KCl   500mM Tris-HCl, pH 7.6100mM Tris-HCl, pH 7.6   100mM MgCl2100mM MgCl2    10mM DTT10mM DTT

• 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.

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'

As they are read on DNA sequencing gels using the Universal primer:

M13mp7.......EcoR1....BamH1.SalI..PstI..SalI..BamH1....EcoR1GGCCAGTGAATTCCCCGGATCCGTCGACCTGCAGGTCGACGGATCCGGGGAATTCM13mp8..........HindIII.PstI.SalI...BamH1.SmaI.EcoRGGCCAGTGCCAAGCTTGGCTGCAGGTCGACGGATCCCCGGGAATTCGTAATCATGM13mp9.......EcoR1.SmaI.BamH1..SalI..PstI..HindIIIGGCCAGTGAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTTGGCGTAATCATGM13mp10...HindIII..PstI..SalI..XbaI..BamH1..SmaI..SstI..EcoR1GCCAAGCTTGGGCTGCAGGTCGACTCTAGAGGATCCCCGGGCGAGCTCGAATTCGM13mp11...EcoR1..SstI..SmaI..BamH1..XbaI..SalI..PstI..HindIIIGTGAATTCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGCCCAAGCTTGGM13mp18HindIII.SphI..PstI..SalI.XbaI.BamH1.SmaI.KpnI.SstI.EcoR1AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCM13mp19EcoR1.SstI.KpnI.SmaI.BamH1.XbaI.SalI.PstI..SphI..HindIIIGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTT