سید طبائی

دارد و آن آگاهی است يك فضیلت وجىد رد جهان تنها است جهل آن و ؛ گناه يك تنها و

مىالان-عارف زبرگ

Genetic engineering Calculations for Molecular Biology and

Biotechnology

Frank H. Stephenson

Molecular cloning A laboratoay manual

Sambrook and Russell

Prefix choice should result in >1 and is < 1000 0.00000005kg = 50 ng no 0.05 ug 0.0025 ml =2.5x 10-3 ml = 2.5ul 0.03 ug = 3x 10-2 ug = 30 ng

Solution W/W: g/g (Lactophenol)

Solution W/V: g /ml(sugar solution)

Solution V/V:ml/ml (alcohol )

Molecular weight (MW) =sum of its atomic weights

Pure /MW with water:

Normal concentration : amount of soluble material g/L

Mol:equivalent to 6.023 x 1 023 molecules

Molarity M: MW(g)/L (exactly 1L)

Molal concentration (m): >1L

The concentration coefficient X

Storage >1X

Working = 1X

Percent solution

Percent of the amount(g/100cc), (cc/100cc)

EX. 200cc Etanol 95%(0,95x200=190 )

190cc absulat Alcohol + 10cc water

Unit volume

1 Liter = 10 dcL =100 cL

100 dc3

Cm3 =cc =ml=(10x10x10) mm3

mm3 =ul

Converting molarity to percent =

Converting percent to molarity=

Normality= M/Capacity MW

Amount at 100 x10

10

M X MW

Working solution prepared

Direct proportion

Increase both or Reduced both (M1V1≠M2V2)

Inverse proportion

Increase one with Reduced Other(M1V1=M2V2)

TE buffer 50 cc (Tris 10 Mm و EDTA 1 Mm )

Stock (Tris 1M ,EDTA o.5M)

Centrifugation

nomogram

Biological materials used for DNA profiling

Blood

Hair

Saliva

Semen

Body tissue cells

vaginal cells

DNA extraction

Step1:Sample preparation:

BLOOD : EDTA (1mg/ml)

Sputum :20 ‘/95oc

Vaginal Swab;

Swap: 2 hours in dry air

In 200 ml of distilled water

30’/56oc and 10’/95oc

Tissue

Methods of physical and mechanical

Grinding: with a mortar and pestle Chinese

Freezing and thawing( membrane failure)

shock thermal

Sonication

chemical (enzymes and some detergents)

Genetic engineering

step2

◦ Disrupt cell

Animal cellular SDS

Plant cellular pectinase /cellulase

Bacteria + lysozyme (peptidoglycan)

Bacteria - lysozyme& EDTA

(lipopolysaccharide)

◦ Buffer(Tris)

Step3

Separation of DNA

RNA

Liped

Glucose

Protein

……

Ion-exhange chromatogaraphy

Enxymatic digestion & organic solvents

DNA Extraction :

Cell lysis buffer(STE)

Tris (10mM,pH 8): buffer

EDTA (1mM): Inhibition of DNase

NaCl 100mM : physiologic

SDS 1%: Dissolve Lipid

Proteinase K:

Endonuclease: peptide bonds in the carboxylate

Aliphatic(linear),(aromatic)ring,hydrophobic

enzyme's stability 4-12.5 (optimal PH is 7.5 – 8).

temperature (20 to 65oc ) optimal 37oc

Concentrations: 50-100 μg/ml

Remove DNases and Rnases

activation of the enzyme: SDS 1%

The removed organic materials:

Phenol + chloroform 1/1

Denaturation of proteins (phenol / pH >7)

DNA&RNA(White layer between the

aqueous phase & phenol) Ribonuclase

remove phenol (Chloroform )

Removed salts

concentrate the DNA:Sodium acetate

The DNA is ionized (Reduced solubility in non-

aqueous media

High purity alcohol :The insoluble DNA

Washing: ethanol 70

Drying, DNA dissolved in distilled water

Nucleic acid quantification

ultraviolet(uv)spectroscopy

Pure DNA A260/A280 ratio close to 1.8.

phenol or protein contamination < 1.8.

RNA is present in the DNA prep >1.8

Pure RNA ratio close to 2.0

Absorbance and optical density (OD)

1 OD of dsDNA = 50ug/mL

1 OD of ssDNA = 33ug/mL

Calculating DNA concentration as a mM mount

Calculate Molarity solution DNA

The Absorbtion coefficient (E260) for a solution 1

mM solution of dsDNA is 6.7

solution of ssDNA is 8.5

resuspended the DNA in a volume of 50 ul.

dilute 20 ul into a total volume of 1000 ul distilled water.

Absorbance 260= 0.550 ; 280=0.324.

DNA concentration of the 50 uL plasmid prep(1.35ug/ul)

What is the A260/280 ratio(0,55/0.324) of the purified

DNA? 1.7

:روش کار

نمونه +µl 500 (ورتکس ونيمساعت در دمای محيط)کننده بافر ليز

µl2 پروتئينازK (20mg/ml)(h1 C /°55 15’و/C °80 )

(.12000دقيقه سانتريفوژ 2ورتکس و)محلول فنل بازي هم حجم

مايع روييDNA(4 اليه, DNAرسوب ، اليه نازک پروتئين، فنل و)

اضافه کردن هم حجم کلروفرم( سانتريفوژ دقيقه 2ورتکس وrpm 2000)

مايع رويي DNA (3 اليه در صورت شفاف نبود مايع مرحله قبل تکرار

(شود

1/10 محلول رويي استات سديمM3 برابر حجم الکل مطلق 2و

10‘سانتريفوژ ,ورتکسC °5 /rpm 12000

دو بار(حذف استات سديم ) 70شستشو با الکل

37/ ’10خشک کردن رسوبoc

آب مقطر استريل اضافه ودر فريز نگهداری شود

sizing DNA fragments by

gelelectrophoresis

POLYMERASE

CHAIN

REACTION

PCR (technique that takes a specific sequence of DNA) fotocopy

Kary Mullis

1983 (PCR was invented )

1985 (The first published)

1993 (the Nobel prize for Chemistry )

DNA

Example of bonding pattern.

Primary strand

CCGAATGGGATGC

GGCTTACCCTACG

Complementary strand

WHAT IS IN THE PCR REACTION ?

Template DNA

Primers

Reaction buffer )Tris, Ammonium and/ or Potassium ions, Magnesium ions, Bovine serum albumin ]

Nucleotides [ dNTPs ]

DNA Polymerase

Primer design (temperature calculation)

Melting temperature (Tm) is temperature at

which the primer will dissociate.

Annealing ) should be just 1-2 C below Tm.(

Both primers should have approximately the same

Tm value.

Tm estimation:

Tm = (4 x [G+C]) + (2 x [A+T])ºC

Preparation Primer

Shelf life of oligonucleotide

• Acidic PH values (Sensitive) MWG-Biotech (PH-8-8/5)

• Nucleases(Danger of degradation)

• suitable hygiene measures at the workplace

– aid by salts two & three-valent ions Low amounts of EDTA

• MWG-Biotech 10-20mM tris- EDTA

• cinnaGen :purest water

• SSDNA(less stable in freezing &thawing)

– Kept to a minimum

– Shorter intervals(1-2days) aliquot at 40c

• SSDNA(less stable in freezing &thawing)

– Kept to a minimum

– Shorter intervals(1-2days) aliquot at 40c

Short-term

◦ Aliquots should be prepared and stored at -200c

is not an adequate for the exclusion of enzymatic reaction

Long-term( >1/2years)

◦ Recommened to store at -800c

◦ Up to two years

OD(optical Density)

MW

Length

Tm(melting temperature)

GC content

Volume for 100 pmoles/ul(uM):

Primer :

Solution( ug)

Lyophilized ( pmol)=

OD Mw Pmol uM/ul(500ul) 50ul work stock(10uM/ul)

6 8201 21948 43.9 11.39

Pmol.ul=uM

Primer work:10 um/ul Necessary amount :./1-1 ul

Standard reaction :./4-./5 ul

Short-term

◦ Aliquots should be prepared and stored at -200c

is not an adequate for the exclusion of enzymatic

reaction

Long-term( >1/2years)

◦ Recommened to store at -800c

◦ Up to two years

What is Taq polymerase?

the DNA polymerase I for Thermus aquaticus

half life : 95oc >40

Many of its enzymes (including DNAP I) will not

denature at high temperatures

PCR

انواع پلی مرازها

حساس به حرارت

Klenow fragment

T4 DNA polymerase

مقاوم به حرارت

Tag polymerase

Ampli tag

Hot start tag

• DNA polymerase • Strings of DNA synthesis on template strings

»dNTP »Primer

• DNA polymerase I :

» Exonucleases activity 3’→ 5` proof reading(Klenow)

» DNA polymerase activity 5`→ 3` »activity RNase H

• Application: • DNA Labeling Nick translation • Created ends Blunt

dNTP MIX

10mM of each (dATP,dCTP,dGTP,dTTP)

PH:7.5

Store:-20oc

Shipment :Wet ice

Final concentration: o.2mM each/ul

.2x100=20 (2ulx/100ul reaction)

Tag DNA polymerase

Concentration :5 unit/ul

Store:-20oc

Shipment :Wet ice

Final concentration: o.15 ul

2.5ul/100ul reaction

Mgcl2 (50mM)

Final concentration: 1.5mM

1.5x100=150 (3ul/100ul reaction)

PCR buffer:10x Final concentration:1x

1x/100ul reaction=100÷10ul =10ul

Typical PCR mix

In a thin wall Eppendorf tube assemble the following

PCR components Amount

Template DNA (5-200 ng) : 1 µg

1 mM dNTPs (200 uM final)

10 X PCR buffer(pH 8.3-8.8)

25 mM MgCl2 (1.5 mM final)

20 uM primer(20 pmoles final)

5 units/uL Taq DNA polymerase(1.5 units)

Water

Final Volume

variable

10 uL

5 uL

3 uL

1 uL

0.3 uL

variable

50 uL

:

Ramp timeزمان بين دو مرحله برای تغييردما درترموسايکلررا گويند

محصول سکانس هدف که درانتهای دارای سکانس پرايمراست: Amply cone

THE BASICS OF PCR CYCLING

30 TO 35 CYCLES EACH COMPRISING OF :

Denaturation ( 94° C ) for 30 seconds

Annealing ( 54-60° C ) for 30 seconds

Extension ( 72° C ) – Time depends on product size

فرايند تقليب) denaturing (

باز شدن دو رشتهDNA با کمک حرارت با شکستن پيوند هيدروژنی

(ثانيه 30اکثرا )درجه 92-100معموال حرارت

فرايند اتصال(Annealing)

اتصال پرايمر به الگو با کاهش درجه حرارت بر اساس قانون واتسون و گريک

درجه کمتر از 3-5دمای اپتيموم معموالTM ثانيه است 30است زمان حدود

فرايند طويل شدن(extention)

گسترش پرايمر بر روی رشته الگو با کمک آنزيم پليمراز در حرارت مناسب

دما آنزيمDNA polymerase حدودoc72 است (1000هرbp حدود يک دقيقه)

PCRمراحل

PCRسيکل در

محصول طول مشخص ندارد و سنتز تا زمان توقف و سيکل بعد ادامه دارد:و دوم اول

(طول مشخص )قطعاتی از توالی بازها از الگوی هدف سنتنز می شود : سيکل سوم

تکثير تصاعدی توالی باز های :سيکل چهارمDNA

محاسبه تعداد کپی ها

(2n -2n)X N= تعداد سيکل هاX=تعداد نسخه از الگوی اوليه

عوامل محدود کننده

سيکل 25-35غير فعال شدن آنزيم بعد از

جزء کلنوDNA polymerase 1 به حرارت حساس و بايد درهرسيکل اضافه شود

افزايش غلظت رشته هدف و رقابت با پرايمر(Reannealing)

PCR Cycles

PCR Cycles

PCR Cycles

PCR Cycles

PCR Cycles

3” 5”

3”

3”

3”

5

”

5”

Enhancers of PCR

Formamide 5%

DMSO <10%

PEG 5-15%

Glycerol 10-15%

Tween20 0.1-2.5%

Conventional PCR

Nested-PCR

RT PCR :(Reverse Transcriptase-PCR)

Real time PCR:

Multiplex-PCR

ARMS-PCR(Amplification Refractory Mutation System)

Touchdown PCR

Asymmetric PCR

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