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核酸和蛋白质吸光度、分子量、摩尔浓度之间的相互换算和引物的稀释   

2009-07-10 11:58:06|  分类: 生物遗传天地 |  标签: |举报 |字号 订阅

 

Nucleic Acids and Protein Calculations and primer dilutions

核酸和蛋白质吸光度、分子量、摩尔浓度之间的相互换算和引物的稀释

Metric Prefixes

Prefix    Symbol    Factor

1kilo (k )= 100centi(c)=1000milli(m)=micro&micro=1000000000nano(n)=1000000000000pico(p)=1000000000000000femto(f)=1000000000000000000atto(a)=1000000000000000000000zepto(z)

Spectrophotometric Conversions

吸光度1A260 unit of double-stranded DNA=50 µg/ml

1 A260 unit of single-stranded DNA=33 µg/ml

1 A260 unit of single-stranded RNA=40 µg/ml

DNA Molar Conversions

1 µg of 1,000 bp DNA =1.52pmol(3.03pmol of ends)

1 µg of pBR322 DNA =0.36pmol DNA

1 pmol of 1,000 bp DNA =0.66 µg

1 pmol of pBR322 DNA =2.8 µg

Formulas for DNA Molar Conversions

For dsDNA(double-stranded DNA 双链DNA)

To convert pmol to µg:

   pmol × N × 660 pg/pmol × 1 µg/106pg = µg

To convert µg to pmol:

   µg × 106pg/1 µg × pmol/660pg × 1/N = pmol

For ssDNA(single stranded DNA)

To convert pmol to µg:

   pmol × N × 330 pg/pmol × 1µg/106pg = µg

To convert µg to pmol:

   µg × 106pg/1 µg × pmol/330pg × 1/N = pmol

where N is the number of nucleotides and 330 pg/pmol is the average MW of a nucleotide

Protein Molar Conversions

100 pmol of 100 kDa protein=10 µg

100 pmol of 50 kDa protein=5 µg

100 pmol of 10 kDa protein=1 µg

100 pmol of 1 kDa protein=100 ng

Protein/DNA Conversions

1 kb DNA=333 amino acids =37 kDa protein

270 b DNA= 10 kDa protein

810 b DNA= 30 kDa protein

1.35 kb DNA= 50 kDa protein

2.7 kb DNA= 100 kDa protein

average MW of an amino acid= 110 daltons

Dalton (Da) is an alternate name for the atomic mass unit, and kilodalton (kDa) is 1,000 daltons. Thus a protein with a mass of 64 kDa has a molecular weight of 64,000 grams per mole

Agarose Gel(%):Resolution of Linear DNA

recommended % Agarose

Optimum Resolution for Linear DNA (Size of fragments in nucleotides;bp)

0.5

1,000-30,000

0.7

800-12,000

1.0

500-10,000

1.2

400-7,000

1.5

200-3,000

2.0

50-2,000

Polyacrylamide Gel(%):Resolution of Protein

Recommended

% Acrylamide

Protein Size Range

8

40-200 kDa

10

21-100 kDa

12

10-40 kDa

Length/M.W. of Common Nucleic Acids.

Nucleic Acid

Number of Nucleotides

Molecular Weight

lambda DNA

48,502(dsDNA)

3.2 × 107

pBR322DNA

4,361(dsDNA)

2.8 × 106

28S rRNA

4,800

1.6 × 106

23S rRNA(E.coli)

2,900

1.0 × 106

18S rRNA

1,900

6.5 × 105

16S rRNA(E.coli)

1,500

5.1 × 105

5S rRNA(E.coli)

120

4.1 × 104

tRNA(E.coli)

75

2.5 × 104

*Molecular weights based on actual sequence.

Stardards

1.  Average MW of dsDNA base pair = 600.

2.  Average MW of ssDNA base =330.

3.  Average MW of RNA base =340.

寡聚体定量:

20-mer,A260 = 1的贮存液含有5 nmol寡聚体:5 nmol = 33 µg/(20×325)

40-mer,A260 = 1的贮存液含有5 nmol寡聚体:2.5 nmol = 33 µg/(40×325)

pmol为单位的引物转换为µg为单位的引物:

(X pmoles×长度bp×325)/ 1,000,000

例:10pmoles的25-mer,则为

(10pmol×25bp×325)/ 1,000,000 = 0.081 µg primer

µg为单位的引物转换为pmol为单位的引物:

(X pmoles×1,000,000)/(长度bp ×325)

例:0.1µg的20-mer,则为

(0.1µg×1,000,000)/(20bp×325)= 15.4 pmoles primer

PCR扩增反应引物浓度的计算:

引物毫摩尔浓度(mM)= pmoles/µl

例1:100µl PCR反应体系中有20pmol的引物 = 0.20 mM

例2:引物为24bp,并溶解于0.1ml µl H2O中;

10µl溶液稀释至1.0ml测定其A260为:A260 = OD260 = 0.76;

则贮存液在260nm(A260)的吸光度为76;

0.1ml的贮存液含有7.6个单位的A260;

引物碱基组成为:A=6, C=6, G=6, T=6;

260nm引物的摩尔消光系数 = a(16,000) + b(12,000) + c(7,000) + d(9,600)

注:a、b、c、d分别是 A's、G's、C's、T's的碱基个数;

PCR引物的摩尔消光系数= 6(16,000) + 6(12,000) + 6(7,000) + 6(9,600) = 267,600

则PCR引物贮存液的摩尔浓度为:76/267,600 = 284 mM

To convert pmol to µg:

pmol × N × 330 pg/pmol × 1µg/106pg = µg

To convert µg to pmol:

µg × 106pg/1 µg × pmol/330pg × 1/N = pmol

where N is the number of nucleotides and 330 pg/pmol is the average MW of a nucleotide

How to Dilute Your Primers

1. Oligo DNA是以OD260单位来计算的,这是指在1ml体积1cm光程标准比色皿中,260nm波长下吸光度为1A260的Oligo溶液定义为1 OD260单位,根据此定义,1 OD260单位相当于33μg的Oligo DNA,您可以根据此数据和您的Oligo DNA分子量,计算得到摩尔数以计算不同摩尔浓度的溶液。

2. 引物序列的分子量计算公式如下:

MW=(A碱基数×312)+(C碱基数×288)+(G碱基数×328)+(T碱基数×303)-61

例如:引物TGGGCGGCGGTTGGTGTTACG   A=1 C=3 G=11 T=6

MW=(1×312)+(3×328)+(6×303)-61=6541

3. Oligo DNA的分子量也可以用以下近似方法计算:Oligo DNA中的每个脱氧核苷酸碱基的平均分子量近似为324.5,则一条Oligo DNA的分子量=碱基数×324.5。

例:您得到一管标为5 OD260的20 mer Oligo DNA

分子量=20×324.5=6490

质量数=5×33=165μg

摩尔数=165/6490=0.025μmol=25nmol

若加灭菌双蒸水400μl溶解,则浓度为25nmol/400μl=62.5μM

4. 装有引物的eppendorf管一般保存于-20℃,临用前稀释。

5. 由于Oligo DNA呈很轻的干膜状附在管壁上,打开时极易散失,所以打开管子前请先离心10,000rpm,1min,然后再慢慢打开管盖。

6.在装有引物的eppendorf管内加入100-500μl双蒸水,盖上管盖,充分上下振荡5-10分钟,再次离心10,000rpm,1min。

7. 计算原引物管primer的浓度(必要时测OD260核对厂家提供引物量是否正确)。

8. 计算并将应用primer稀释为10pmol/μl。

9. 标明原引物管、应用引物管、稀释方法,置-20℃保存

干粉引物溶解稀释方法:

接到引物后,在开启离心试管之前最好在3000-4000Rep/min的转速下离心1min,以防开盖时引物干粉散失。

I. 稀释成100pmol/ul   1OD 需加水量(ul)=1OD相当于umol 数×10000

     例如,您拿到的引物DNA合成报告单上标有1OD≈0.0035umol ,包装量是1OD/管,如果您希望将引物浓度定为100umol/L,那么只需用35ul无核酸酶的双蒸水将1OD的引物干粉溶解即可。

II.  The information like this:

For example:

MW: 6428.98

Pmol: 10000

Volume: 0 u L

10000Pmol convert to µg

pmol×10000×6824.98pg/pmol×1µg/106pg

=68249800µg /106

=68.2498µg

=0.068mg

Primer Dilution

I have got the labeled primer information like as follows:

Name: AAG01F

Sequence: NED GCT TTT GAT CAA TCG CCC AA(20mer)

MW: 6428.98

Pmol: 10000

Volume: 0 µL

So, It convert pmol to µg is:

10000Pmol convert to µg

pmol×10000×6824.98pg/pmol×1µg/106pg

=68249800µg /106

=68.2498µg

=0.068mg

And, first we want to dilute labeled primer to 100µM:

10000pmol/xµL(TLE,TE or H2O)= 100µM

10000pmol/xµL(TLE,TE or H2O)=0.1nM

10nmol/xµL(TLE,TE or H2O)=0.1nM

X=100µL

III. If you have a primer in solution that is 100 micromolar that is the same as 100 nmol/ml or 100 pmol/µl We only need 12 picomoles in one sequencing reaction so dilute in the following method: Do a 1:10 dilution, take 10 µl of 100 µM solution add 90 µl of water. This will give you a solution of 10 pmol/µl. Add 1.2 µl of primer to the sequencing mix you supply to Core Labs to provide us with 12 picomoles

V. If you have primer information likes this: 56.8nm, which is mean 56.8nmol. So, if you want to dilute to 100µM just add 568µL TLE,TE or ddH2O, vortex, and save into -80℃。The working stock primer was 10µM, take 10 µl of 100 µM solutions add 90 µl of TLE, TE or water. And the working primer maybe was 10µM, 5µM or 2µM depend on different PCR reactions.

VI. Liquid primer dilution as follow format:

The final concentration(pmol/ul) =original concentration(pmol/ul)×volume of original primer(ul)÷(add volume of original(ul)+volume of water(ul))

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