重组DNA的分离、克隆与测序实验手册

Units and formulas

Units:

1 mg = 10-3 g.
1 ug = 10-6 g.
1 ng = 10-9 g.
1 pg = 10-12 g.

1 kb of DNA	= 6.5 x10+5 Daltons of duplex DNA (sodium salt)
	= 3.3 x10+5 Daltons of single-stranded DNA (sodium salt)
	= 3.4 x10+5 Daltons of single-stranded RNA (sodium salt)

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

1. 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

2. 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

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.

Codon chart and amino acid symbols

                      Second base

One and three-letter 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

Biomek configuration for single stranded DNA isolation

Consensus sequences in nucleic acids