Bone Marrow Engraftment Confirmation
Qiagen QIAamp DNA Blood Mini Kit (#51104)
ETOH (96-100%)
SRY primers (see last page)
Green Mix (Promega PRM7122)
Agarose (LifeTech Invitrogen 16500500)
TAE buffer
SYBRsafe gel stain (LifeTech Invitrogen S33102)
200L whole blood/sample
DNA Reagent and Sample Preparation:
1.
2.
3.
Turn on a heating block and set it to 56oC.
Add 1.2mL protease solvent to the vial containing lyophilized Qiagen Protease. Place on ice
or at 4o. Shake Buffer AL thoroughly to mix; check for precipitate. If present, dissolve it by
incubating Buffer AL at 56oC. Add ETOH to Buffer AW1. Add ETOH to Buffer AW2. If sample
volumes are smaller than 200L, bring them up to this volume using PBS.
If desired, prepare buffy coat from whole blood samples. Centrifuge whole blood at 2500 x g
for 10 minutes at room temperature. After separation by centrifugation, the buffy coat is the
intermediate layer. This yields approximately 5-10 times more DNA than an equivalent
volume of whole blood.
DNA Isolation Procedure:
1. Pipet 20L Qiagen Protease into a 1.5mL microcentrifuge tube.
2. Add 200L whole blood (or buffy coat).
3. Add 200L Buffer AL. Pulse vortex for 15 seconds.
4. Incubate at 56oC for 10 minutes.
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Short spin samples in centrifuge to remove droplets from lid.
Add 200L ETOH. Pulse vortex for 15 seconds.
Short-spin samples in centrifuge to remove droplets from lid and sides of tube.
Place QIAamp Mini spin columns into 2mL collection tubes.
Carefully apply samples from step 7 to columns from step 8, without wetting the rims. Close
the caps.
Centrifuge at room temperature at 6000 x g for 1 minute.
Place the columns into clean 2mL collection tubes and discard the tubes containing the
filtrate. (This filtrate is not compatible with bleach; do not add bleach!)
Carefully add 500L Buffer AW1, without wetting the rims. Close the caps.
Centrifuge at 6000 x g for 1 minute.
Place the columns into clean 2mL collection tubes and discard collection tubes containing
filtrate.
Add 500L Buffer AW2 without wetting rims. Close the caps.
Centrifuge at full speed, 20,000 x g for 3 minutes.
Place columns into new 2mL collection tubes (not provided) and discard collection tubes
containing filtrate.
Centrifuge at full speed for 1 minute.
Place columns into new 1.5mL microcentrifuge tubes (not provided) and discard collection
tubes containing filtrate.
Add 200L Buffer AE (preferred for eventual storage) or distilled water. Let stand at room
temperature for 1-5 minutes.
Centrifuge at 6000 x g for 1 minute. Discard columns and label tubes appropriately. This
process should yield 4-12 g of DNA (from 200L human blood).
RO’R 2013, January
PCR:
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Calculate components required; SRY utilizes 2 primers: forward and reverse.
Clean surfaces with ETOH (let dry COMPLETELY).
Place green mix, water, and primer aliquots on ice.
Dilute SRY primers with ddH2O to 50M, if necessary. DO NOT vortex primers; only flick
lightly! (If primers were supplied by Fisher, the stock concentration is likely 200M, so
merely dilute 1:4 in ddH2O water.) 200M stock is at -20o, box 20; 50M dilution is at -20o,
box 16.
Make master mix according to calculations from step 1. DO NOT vortex! Place on ice.
Label small PCR tubes (depending on which thermal cycler will be used), including a positive
(male) and negative (female) control.
Divide master mix among tubes, at 24L each.
Add 1L DNA to the mix in each tube.
Close lids tightly and place in thermal cycler. Use protocol S, vessel type “thick”, volume
25L, and heated lid “yes”. This process will take ~ 3 hours. In the meantime, prepare the
gel.
Gel Preparation:
1.
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5.
Assemble gel apparatus. Gaskets will fit tightly.
Combine 90mL ddH2O with 10mL 10x TAE buffer and 2g Agarose in a clear Erlenmeyer flask.
Microwave for ~ 1 minute+45 seconds or until transparent (no crystals are visible).
Add 10L SYBRsafe; swirl slowly so as not to create bubbles.
Pour into mold and add comb (thick side down) immediately. Use a yellow tip to move air
bubbles to edges. Let stand for 40 minutes to 2 hours. If gel will not be used within 2 hours,
store in buffer up to 24 hours at room temperature.
6. Combine 720mL ddH2O with 80mL 10x TAE in a graduated cylinder. This will yield 800mL of
buffer; 800mL of buffer is needed for each gel box.
7. When gel is ready, gently lift center section of box out and rotate it so that the wells are
toward the black end, indicating the direction of the current. This will fit rather loosely.
8. Pour all of the running buffer from step 6 over the gel and into the apparatus.
9. DNA ladder samples are stored at 4o. Load 3L of ladder and 10L of each sample, including
appropriate controls.
10. Run at 150V for ~ 1 hour. (No need to increase voltage when connecting more gels to the
system.)
11. Turn on imager prior to exposure to allow time to cool.
SRY Sequences:
Forward: TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA
Reverse: CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA
RO’R 2013, January