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Manual Extraction of DNA from The Blood

Manual Extraction of DNA from The Blood. Prepared by: Dr. Abbasi. - Blood Sample. - Distilled water. - Ice and Plastic trey. Materials. Auto clave. - PH meter. - Balance. - - Micro pipette (transfer pipette) (2-20µl, 10-100µl, 100-1000µl) Centrifuge. - Shaking Water bath. -

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Manual Extraction of DNA from The Blood

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  1. Manual Extraction of DNA from The Blood Prepared by: Dr. Abbasi

  2. -Blood Sample. - Distilled water. - Ice and Plastic trey. Materials

  3. Auto clave.- PH meter.- Balance.- - Micro pipette (transfer pipette) (2-20µl, 10-100µl, 100-1000µl) Centrifuge.- Shaking Water bath. - Vortex.- Spectrophotometer.- Equipment

  4. - Beakers (50ml, 80ml, 100ml, 500ml). Volumetric flask (100ml, 250ml, 500ml, 1000ml).- - Plastic and glass centrifuge tubes (15ml, 50ml). - Measuring cylinders (50ml, 100ml, 500ml). - Pasteur pipettes. - Pipettes (1ml, 5ml, 10ml). - Eppendorf tube. - Tips with different size. Racks.- Quarts cuvettes. - 15ml 50ml Glassware

  5. Proteinase K (20mg/ml).- Lysis buffer (2X). - SDS 10%. - Salt/ EDTA.- - Chloroform: Isoamyl alcohol. EDTA 0.5M. - Ethanol 99-100%.- 10mM Tris, 1mM EDTA.- 1M Tris PH= 7.6- - TE buffer 10:1 Phenol.- Reagent

  6. 0.1 ml = 100µl 1 ml = 1000µl 100 ml = 10000µl Calculations

  7. The DNA was extracted manually from blood sample. - This method uses SDS- proteinase K method which dissolve the sample and digest the protein component without affecting the DNA. Extraction and purification of DNA

  8. Add 5ml of blood + 45ml of Lysis buffer (2X) to 50ml capped centrifuge tube. Mix the samples using the Vortex for 10min. Put the tubes in the Centrifuge for 10min at 3000rpm. There will be 3 layers Extraction and purification of DNA Procedure

  9. Discard the supernatant. Add 3ml of EDTA salt buffer + 0.3 ml of 10%SDS + 0.1ml of proteinase K to the pellet. Incubate all the tubes over night at 37ºC in shaking water bath. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure

  10. Add 3ml of liquid phenol to each tube. Mix the samples using the Vortex for 10min Put the tubes in the Centrifuge for 10min at 2000rpm. There will be tow layers. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure

  11. Add 3ml of chloroform:Isoamyl alcohol to the upper aqueous phase. Put the tubes in the centrifuge for 5 min at 2000rpm. Tow layers will appear. Add 6ml of ethanol to each tube to precipitate the DNA. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure

  12. Put the tubes up side down until the precipitated DNA is completely dry. Add 0.5 ml of 10mM EDTA buffer in 2 ml eppendorf tube to redissolve the DNA over night ad store in -20 degree C. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure

  13. Dilute 25µl of DNA sample with 2ml of distilled water in quartz cuvette and mix throughly. The concentration of DNA sample was assessed by using spectrophotometer. The optical density was recorded at 260 and 280nm. The 260/280nm absorbance ratio was calculated. Measure of DNA concentration

  14. Extraction of DNA from whole bloodProcedure • 1. Pre-warm kit to room temperature before use. - Pre – warm Lysis solution by placing in 37oC for 20 min and softly shake. • 2. Mix 700µl of sample with 400 µl of Lysis Solution (for whole blood) and vortex 15-20 sec. (The sample should be completely homogenous suspension at this step. Any aggregation, clot or insoluble materials could be degraded by softly pipetting or removed). • 3. Add 500 µl of Precipitation Solution (For whole blood ) mix by vortexig 3-5 seconds, then centrifuge 12,000 g for 10min. • 4. Decant by gently inverting of tube and placing the tube on tissue paper for 2-3 sec. down ward. Care for avoid of cross – contamination between different samples.

  15. 5. Add 1 ml Wash Buffer to pellet, mix by 3-5 seconds vortexing and centrifuge at 12,000 g for 5min, then decant (for whole blood and tissue sample repeat this step once more). • 6. Pour off the Wash Buffer completely and dry pellet at 65oC for 5 min. (up to dry). • 7. Suspend pellet in 50µl of Solvent Buffer (for serum or plasma sample, suspend pellet in 30µl) by gentle shaking and placing at 65oC for 5 min. Wash the wall of tube for mixing of any residual pellet by softly pippeting. • 8. Precipitate unsolved materials by centrifuge, 30 sec at 12,000 g, supernatant contains purified DNA. • 9.Measure DNA concentration spectrophotometrically or visually after electrophoresis in fresh 1 % agarose gel.

  16. Easy DNA extraction Materials Easy DNA extraction Materials • What You Need • Frozen Banana • Table Salt • Warm water • Liquid soap • Blender • Toothpicks • Strainer (Coffee filter ,#2 or # 6) • Glass jar • Cold 95 % cold ethanol • spoon • Wooden stir stick • Knife

  17. DNA Extraction • * Extract DNA: • 1-Peel the banana and break or cut it into small pieces. • 2-Put the banana in the blender and cover them with warm (not hot) water. • 3-Add a teaspoon of salt to the mixture. • 4- Blend the mixture until it is a thick liquid and will pour easily. about 10 seconds.

  18. DNA Extraction • 5-Pour the mixture through the strainer and into the glass jar about halfway. • 6-Add 2 teaspoons of liquid soap to the banana pulp in the glass jar. • 7- Stir the mixture very gently. • 8-Pour very cold alcohol (in the freezer for about 30 minutes) slowly down the side of the jar into the mixture. • 9- You don't need much alcohol, just enough to form a thin layer on top of the banana mash.

  19. DNA Extraction • 10-Allow the mixture to sit undisturbed for 5 or 10 minutes. • 11-The DNA will coalesce in a layer, most likely between the banana mash and the alcohol. • 12- Use a toothpick or unbent paperclip to gently pick up the long, stringy strands of DNA. This is not a single molecule, of course, but a combination of billions of DNA molecules extracted from the banana cells.

  20. !!Result

  21. Any Question? Thank You

  22. استخراج DNA از خون به روش فنل-کلروفرم

  23. وسایل و مواد لازم • 1- 200 میکرو لیتر خون وریدی • 2-دو عدد میکرو تیوپ • 3- سمپلر و سر سمپلر • 4-فنل و کلرو فرم • 5- ایزو پرو پانل • 6-ظرف حاوی یخ • 7- اتانل 70% • 8-سانتریفیوژ یخچالدار • 9-آب مقطر • 10- دستکش یک بار مصرف

  24. استخراج DNA • 1-در یک میکروتیوپ 200میکرولیتر خون +EDTA می ریزیم • 2-100 میکرولیتر فنل و 100 میکرولیتر کلروفرم(هم حجم خون) به آن می افزائیم. • 3-یک دقیقه ورتکس میکنیم • 4-5 دقیقه در دمای اطاق نگهداری می شود • 5-سپس به مدت سه دقیقه با سرعت 13000 دور در دقیقه و دمای ºc 4 درجه سانتریفیوژ می گردد. • 6-پس از سانتریفیوژ سه فاز قابل تشخیص است. از بالا: فاز آبی حاوی DNA ، فاز پروتئینی و فاز آلی حاوی غشائ لیپیدی وRNA

  25. DNA extraction with phenol/chloroform aqueous top phase contains the majority of DNA, interphase mostly proteins, and lower organic phase most of the RNA and lipids

  26. استخراج DNA • 7-فاز آبی را با دقت با یک سمپلر جدا کرده داخل یک میکروتیوپ ( 1/5ml(جدید منتقل می کنیم. • 8-هم حجم فاز آبی ایزوپروپانل سرد می افزائیم • 9- مخلوط را به خوبی هم زده (ورتکس می کنیم) و سپس 15دقیقه روی یخ قرار می دهیم. • 10- سپس به مدت 11 دقیقه با سرعت 13000 دور در دقیقه در دمای ºc 4 سانتریفیوژ می کنیم. • 11- محلول روئی را به آهستگی خالی کردهو به روسوب ته تیوپ 0/5 میلی لیتر اتانل 70% سرد اضافه می کنیم.

  27. استخراج DNA

  28. استخراج DNA • 12-سپس به مدت 5 دقیقه با سرعت 12000 دور در دقیقه در دمای ºc 4 سانتریفیوژ می کنیم. • 13- محلول روی را جدا کرده(حاوی RNA) و اجازه می دهیم رسوب (حاوی DNA) در دمای آزمایشگاه خشک شود. • 14- رسوب را که حاوی DNA است با 20 میکرو لیتر آب مقطر حل می کنیم. • 15- نام گروه را روی تیوپ بنویسید و در 20- درجه در فریزر نگهداری کنید. • جهت رویت روی ژل آگارز run کرده پس از رنگ آمیزی در ژل داک مشاهده می نمائیم.و جهت خواندن درجه خلوص و تعیین غلظت DNA از دستگاه اسپکترو فتومتر و خواندن استفاده می کنیمOD

  29. Basic PCR protocol • Add the following components to a sterile 0.5ml micro-centrifuge tube sitting on ice: • Components Volume Final Con. • Autocl. Distill. water to14 µl • 10 X PCR buffer 2.5 µl 0.2mM each • 10mM dNTP mix 0.5 µl 0.2 mM each • 50mM MgCl2 0.75µl 1.5mM • Forward primer 1µl 0.5µM • Reverse primer 1µl 0.5µM • Control DNA (4pg/µl) 5µl 20pg • Taq DNA polymerase 0.2 µl 1 unit • Mineral Oil 25µl

  30. Steps Temperature Range of duration (seconds) Min. Max. • Denature 120 • Denature 93°C 10” 45” (30”) • Anneal 40-65°C 10” 60” (30”) • Extend 72°C 20” 90” (60”) • 35 Cycle

  31. Result analysis: • Analyze 10µl of amplified samples in a 2% agarose gel after adding loading buffer. • The presence of 620bp fragment indicates of positive

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