DNA extraction and ampli cation

-HLA-DRB1 gene

• in ependorf tube 200 microL of blood

• add 20 microL protease (powder+bu er then aliquoting), to break down the proteins, our aim is
to get rid of everything except for WBC

• add 200 microL lysing by er (AL), in order to lyse the RBC . more expensive, rapid and
accurate (in hospitals they do not use them, instead they use house kit, cost e ective but not
very accurate)

• vortex for 15 seconds

• water bath for 56 degree for 15 minutes (this is the working temperature of protease)

• add 200 microL pure ethanol to precipitate DNA and RNA

• quick spin to allow proper mixing, everything on the walls of tube will go back in solution

• DNA PURIFICATION

◦spin column tube

◦pipette the blood from eppendorf tube (700 microL) into the spin column tube

◦centrifuge for 1 min at 8000 rpm

◦DNA will reside on lter

◦everything else will be pass the lter

◦add AW1 wash 500 micrL and centrifuge. then discard tube and transfer lter to another
collection tube.

‣ add 25 mL to AW1 bottle before usage.

◦add AW2 wash 500 micrL and centrifuge. discard tube and transfer lter to a third
collection tube

◦centrifuge at full speed the last collection tube to allow ethanol and excess bu er to elute
because ethanol interferes with PCR

◦transfer lter to eppendorf tube

◦add 50 microL AE (Elution Bu er, water RNAase and DNAase free) and incubate for 5
minutes allowing AE to react with DNA and purify it then centrifuge for 5 minutes

◦ discard the top tube

◦NANO DROP (spectrophotometry)

‣ allows to read the concentration of DNA

‣ every concetration above 60 micrograms/mL is acceptable

‣ pure DNA ratio is 1.8

• above means contamination with lipids

• below means contamination with RNA

‣ pure RNA ratio is 2

‣ add 2 microL (1drop) of AE as blank (zeroing machine)

‣ pipette 2 microL of sample into machine and run machine

‣ 74

• PCR ROOM

◦put tube in ice to preserve DNA

◦all Volumes are obtaines following CiVi=CfVf

◦[MgCl2]i=

◦Vi=????

◦[ ]f=

◦Vf=20 microL

◦following CiVi=CfVf, we obtain Vi=

◦PCR mix will be composed of:

‣ Taq bu er 2 microL

 • pipette them on wall

‣ DNTPS 2 microL

‣ MgCl2 1.2 microL

• cofactor of Taq polymerase

‣ Taq polymerase 0.5 microL

• works at 72 degrees C

‣ primers 0.6 microL+0.6 microL

• forward and reverse

• speci c for the gene in research

‣ DNA 5 microL relative to [ ]

• if [ ] around 40, add 8microL of DNA

‣ water 5 microL

◦Vortex and quick spin

◦cycle PCR

‣ 90 C denaturation

‣ 50 C annealing

‣ 70 C transcription

‣ machine rests at 4 degree C once done

Gel electrophoresis

• 2 g agarose

• 5X TBE ( i need 1X, so i take 100 mL and dilute to 500mL) then take 100 mL and mix with 2 g
agarose and microwave for 2 minutes

• add 2 microL ethidium bromide (sensitive to light)

◦ uorescent dye to allow visualization under UV

• Prepare apparatus

◦leave to solidify

◦from minus to positive

◦add TBE bu er

‣ allows current to pass

◦On para n paper

‣ Add 2 microL ladder

‣ Add 2 microL 6X DNA loading

• colors the DNA

• for visibility only

‣ mix and pipette the now 4 microL into the rst well

◦10 microL DNA + 2 microL 6X

‣ pipette into second well

◦do the same for all samples

-3 separated room (wall separated) free of contamination and highly hygienic, each had its
utensils

1. extraction room

• dirtiest room

• iza sar contamination, akhtar chi.

2. PCR room

• cleanest room

3. Post ampli cation room.

• gel electrophoresis.

• NO heparin tube!!! because heparin inhibits the primers in PCR, annealing also is a ected.

• If leukemia patient; specimen is from bone marrow and we cannot take another sample so we
MUST run the sample.

• add RPMI or PBS reagents, 5 min, centrifugation, disregard the supernatent

• 3 to 4 times to completely clean the blood from heparin

• however this will a ect blood volume and components

• inform the Dr.

What is also done in molecular lab?

• Translocation

• HLA typing for siblings (for bone marrow transplant)

• virus qualitative and quantitative analysis.

• all leukemia diagnosis

• UV machine to read electrophoresis

• PCR machine (qualitative)

• RT-PCR (quantitative)

RNA extraction

GAPDH house keeping gene

• Must work at 4 degree C because RNA is sensitive

• in eppendorf

◦250 microL blood

◦1250 microL EL (erythrocytes lysis)

• vortex

• leave in ice for 10 minutes allowing for reagent to work

• centrifuge for 10 minutes at 8000 rpm

• discard supernatant

• add 500 microL of EL

◦Vortex, mix and directly centrifuge for 10 minutes

◦This step is to further clean the sample from RBC and other cells allowing for a better RNA
extraction

• discard supernatant

• add 350 microL RLT

◦Lysing bu er

◦protein destruction

• vortex

• pipette the sample into shredder tube

◦homogenizes solution

◦on lter: debris and waste

◦in ltrate: homogenized pure RNA solution

• centrifuge at maximum speed for 2 min

• discard lter

• add 350 microL 70% ethanol

◦RNA precipitation

• pipette the 700 microL solution into spin column tube

• centrifuge at 8000 rpm for 1 minute (15s)

• discard the ltrate

• transfer lter to a collection tube

• add 700 microL RW1(no ethanol)

◦wash bu er

• centrifuge 8000 rpm 1 min

• transfer lter to another collection tube

• Add 500 microL RPE (+ethanol)

• centrifuge 8000 rpm 1 min

• discard , transfer

• add 500 microL RPE

◦3rd and nal wash

• centrifuge full speed 3 minutes

• discard and transfer to the 4th collection tube

• centrifuge WITHOUT adding bu er

◦allows total puri cation from ethanol because it inhibits PCR

• discard and transfer lter to eppendorf tube

• add 30 microL Elution bu er RNAase free water

• centrifugation 8000 rpm 2 minutes

• discard the lter

◦ solution now contains pure RNA

• NANODROP

◦Ratio reading (pure RNA ratio is 2)

◦1.89

RTPCR Reverse Transcriptase PCR

• 10 microL into PCR tube

• 1 microL oligoDT

• 4 microL 5XD

• 1 microL ribolock RNAase inhibitor enzyme

◦inhibits RNA degradation

• 2 microL DNTPs

• 1 microL RT (reverse transcriptase)

• RTPCR

◦42 degree

◦70 degree

◦(no annealing, no extension)

• run 3 controls

◦pos

◦neg (non template control) no RNA, water instead, no Band, for QC

◦RT minus

‣ proves if RNA pure or not

‣ no RT

‣ no band

‣ if band, this means there is genomic DNA contamination

PCR

• now that our cDNA is ready, we prepare our sample for PCR

• in new PCR tube

◦2 microL cDNA

◦5 microL 10X bu er

◦1 microL DNTPS

◦3 microL MgCl2

◦1.5 microL forward primer

◦1.5 microL reverse primer

◦0.5 microL Taq pol

◦35.5 mL water