Blood RT-PCR

1. 0.9% polysorbate 20 (Tween-20), 5% PEG 6000

Chinese

2. L-Carnitine inner salt (C0158), D-(+)-trehalose dehydrate (T9531), betaine (B2629), heparin (H3393), and Nonidet P-40 (NP-40)

0.6 mM D-(+)-trehalose, 0.48 mM L-carnitine, and 0.8% NP-40.

Extraction of DNA from Blood, protocol 1

Place  paper disc  in a tube containing 20 µL of TE buffer (10 mM Tris-HCl (pH8.0), 0.1 mM EDTA)  or a PBS-based elution buffer containing 0.05% Tween 20 and 0.05% sodium azide

Heat the tubes for 15 min at 50°C

Press the disc gently to the bottom of the tube several times using a new pipette tip for each disk

Heat for 15 min at 97 °C.

Centrifuge the tubes  at 15,000 rpm for 5 min

Take 1∼8 µL of the supernatant (5%∼40% blood eluent) for PCR in the 20-µL PCR reaction.

PCR with KOD FX: 94 oC 2 ', 35 cycles of (98 oC 10, 55 oC 30, 65 oC 45), 68 oC 30`

Extract DNA from  Whatman S&S 903 

Spot 50 ul blood on each on a Whatman S&S 903 card allow to dry for 4 h at room temperature. Place filter paper punch in a microcentrifuge tube Soak in 65 μL of TE buffer Incubate at 50°C for 15 minutes. Press the punches to at the bottom of the tube several times, using a new pipette tip for each punch Heat at 97°C for 15 minutes to elute DNA

FTA Purification Reagent (Fitzco, Inc) 500 ml:

0.29 g NaCl;

5 ml 1 M Tris pH 7.5

1 ml 0.5 M EDTA

2.5 ml Triton X-100

RBC lysis buffer: 1X  ACK (Ammonium Chloride Potassium)

            155mM NH4Cl                      8.29g

            10mM KHCO3                        1.0g

            0.1mM EDTA                        0.2 ml (500mM) 

10X RBC lysis buffer
NH4Cl 80.2 g
NaHCo3 sodium bicarbonate 8.4 g
EDTA disodium 3.7 g
Add water to 1 L, store at 4 oC for 6 months

5 ul of whole blood one can expect between 35,000 and 50,000 white blood cells; each cell containing approximately 7 pg of genomic DNA Taking the upper limit, 350 ng of total genomic DNA is expected from 5 ul of whole blood.

Preparing an FTA Disc for DNA Analysis

1. Take two sample discs from the dried spot For plant samples a 2.0mm disc is recommended.

2. Place discs in the bottom of a spin basket insert.

3. Place the spin basket into a 2.0mL micro centrifuge tube.

4. Add 500µL FTA purification reagent to the basket.

5. Incubate for 1 minute at room temperature.

6. Centrifuge at 6000 x g for 30 seconds.

7. Remove the basket and decant used reagent.

8. Return the basket to the micro centrifuge tube and repeat Steps 4-7 twice for a total of 3 washes with the FTA purification reagent.

9. Add 495µL FTA purification reagent and 5µL Proteinase K (10mg/mL) to the FTA discs in the basket.

10. Incubate for 1 hour at 60°C to remove residual Histones.

11. Centrifuge at 6000 x g for 30 seconds.

12. Remove the basket and decant used reagent/buffer.

13. Return the basket to the micro centrifuge tube.

14. Add 500µl of TE-1 buffer to the tube.

15. Centrifuge at 6000 x g for 30 seconds.

16. Remove the basket and decant used buffer.

17. Return the basket to the micro centrifuge tube.

18. Repeat steps 14-17 twice for a total of 3 washes with TE-1 buffer.

19. Transfer discs to a new tube.

20. Ensure that all the liquid has been removed before performing analysis.

TE Buffer (10 mM Tris-HCL, 0.1 mM EDTA, pH 8.0) Solution 1: 0.1N NaOH, 0.3mM EDTA, pH 13.0 Solution 2: 0.1M Tris-HCL, pH 7.0

Procedure for wash protocol

1. Take a 6 mm punch using a regular single-hole paper punch from a dry spot and place in a 1.5 mL microtube. To ensure that there is no cross-contamination, rinse the cutting end of the punch with ethanol and let it dry between samples.

2. Add 1000 μl of FTA Purification Reagent to microtube and flash vortex 5 times or manually to mix.

3. Incubate for 5 minutes at room temperature (tube may be given moderate manual mixing or vortex if desired).

4. Remove and discard all spent FTA Purification Reagent using a pipette.

5. Repeat steps 2-4 twice, for a total of three (3) washes with FTA Purification Reagent.

6. Add 1000 μl of TE Buffer.

7. Incubate for 5 minutes at room temperature.

8. Remove and discard all spent TE Buffer with a pipette.

9. Repeat steps 6-8 twice for a total of three (3) washes with TE Buffer (punch should look white or pale with most of the blood removed).

2. Incubate 5 minutes at 65oC (deviation from FTA company protocol which incubates at room temperature).

3. Add 260 μl of Solution 2 and flash vortex 5 times to mix.

4. Incubate 10 minutes at room temperature.

5. Flash vortex 10 times.

6. Remove punch and squeeze to recover maximum volume of elute (can use a clean pipette tip to remove punch).

Eluate contains gDNA in TE (66mM Tris-HCL, 0.1nm EDTA). Use 0.5 μl for a 25 μl PCR reaction.

Extraction of DNA using Chelex

Chelex 100 Resin (Cat no. 143-2832, Bio Rad) .

Cut out blood filter

Incubate overnight Transfer 200 μl of the eluate, which contained all soluble elements including antibodies, to a new plate and store at −80°C until use in ELISA.

Aspirate the remaining saponin solution

Add Washing solution to each well at 4°C.

Incubate the plate were horizontally on a shaker for one hour

Aspirate and discarde PBS Add 150 μl of a 6% Chelex in DNase/ RNase free water solution to each well.

Seal the plate using adhesive foil mats (Axygen Biosciences, CA, USA)

Incubate in a water bath for 3*10 minutes at 97°C.

Between 10 minute incubations plates should be briefly centrifuged in order to relieve pressure and ensure optimal DNA elution.

After the last incubation, spin plates at maximum speed for 5 minutes to allow the Chelex to settle.

Remove 120 μl of the DNA containing solution and aliquoted into new plates.

Store samples at −80°C until further DNA analyses.

Also include positive and negative controls

Extraction of DNA (Recommended Protocol)

using Grade 903 and Ahlstrom Grade 226

This protocol is recommended if you need a large amount of DNA. If your testing does not require a lot of DNA, then typically you can use one 3 mm punch and follow the protocol on page 1.

1) Punch 3 circles from the centre of a blood spot using a 3.0 mm paper punch into a sterile 1.5 mL microfuge tube. To prevent cross contamination between samples, punch a clean piece of filter paper 3 times after punching out each sample.

2) Wash the punched circles by adding 500 µL of sterile water to the tube and pulse vortex 3 times, for a total of 5 seconds. Please note: If the wash is pink, the blood sample was not completely dried. Discard these punches and thoroughly dry the remaining blood spots before re-punching the card and repeating step 2.

3) Using a sterile pipette remove excess wash water from the tube and gently squeeze the circles against the side of the tube to remove as much water as possible.

4) Add 100 µL of sterile water to the tube. Ensure the circles are completely immersed in the water by briefly pulse centrifuging the tube for 5 seconds.

5) Transfer the tube to a heating block at 95oC-100 oC for 15-30 minutes.

6) At the end of the incubation period remove the sample from the block and pulse vortex the sample approximately 60 times.

7) Briefly centrifuge to separate the paper punch from the eluant. The eluant now contains the extracted DNA.

8) Using a sterile pipette tip gently remove the spent blood spot circles from the tube and discard them into a biohazard container.

9) Store the eluted DNA at –20oC until required.