LaboPassTMTissue miniProtocol BookLaboPass TM Tissue Mini
Introduction
LaboPass TM Tissue Kit provides a simple and fast method for the isolation oftotal DNA from up to 40 mg of animal tissue or 5 x 106 cultured cells. The
purified DNA using this kit is ready for applications such as PCR, Southernblotting, and restriction enzyme assay. Added Buffer RL (Optional!) toLaboPass TM Tissue Kit also provides additional preparation from up to 1 ml of
mammalian whole blood.

Storage Conditions
All components of LaboPass TM Tissue Kit, except Proteinase K solution,should be stored in room temperature(15-25℃). Proteinase K solution (20mg/ml) can be stored for 2 months at 4℃ without any decrease in activity. Butstorage at -20℃ with usable aliquots is recommended for prolonged
preservation. Under cool ambient conditions, a precipitate may form in buffer
TB. In such a case, heat the bottle at 60℃ to dissolve. LaboPass TM Tissue Kit
is guaranteed for 1 year after delivered to an end user.

Sample amount and expected yields
LaboPass TM Tissue kit is designed for preparation from small amount of tissue.
For most animal tissues, it is recommended the starting amount of sample
should not be over 20 mg per each prep. If it exceeds about 25-30 mg
depending on the tissue type, the efficiency of DNA recovery will besignificantly reduced. We recommend you start with smaller sample amount,and depending on the yield obtained, increase the sample size in subsequentpreparations.
The yields and recommended maximum weights of starting materials forvarious sources are listed below.

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Kit Contents
Cat. No.
No. of preparation
Spin column
Collection tube
Buffer TL
Buffer TB
Buffer BW(conc.)
Buffer NW(conc.)
Buffer AE
Proteinase K solution(20mg/ml)

CME0111
50
50
150
15ml
24ml
19.6ml10.5ml15ml
1.1ml

CME0112
200
200
600
60ml
100ml
76.1ml32.3ml60ml
1.1mlx4

Optional! Buffer RL(1)

60ml

220ml

CME0115
1000
1000
3000
60mlx5100mlx5
76.1mlx5
32.3mlx5
60mlx5
1.1mlx20(1) Optional! Buffer AL is available to purchase.
* All Solution (Buffer) is available to purchase separately.

Little copurification with RNA
LaboPass TM Tissue spin column has very stronger affinity to DNA than RNA.
Although RNase is not treated, the percentage of RNA from total prepared
nucleic acid will be very low even with a transcriptionally active tissue, such asliver or pancreas.
However, if RNA-free DNA is required, RNase should be treated.

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Protocols for Animal Tissue
Before experiment- Prepare the water bath to 56℃- Equilibrate buffer AE to room temperature- Add appropriate volume of absolute ethanol to Buffer BW and NW before first use.
- All centrifugation should be performed at room temperature.
- Mix Buffer TL and TB thoroughly by shaking before use.

- Buffer TB may precipitate at cool ambient temperature. If so, dissolve it in 60℃water bath.

1. Homogenize up to 20 mg of tissue as described in step 1A, 1B, or1C.
For spleen tissue, up to 10 mg can be processed. If the sample is larger than
20 mg (if spleen, 10mg), increase the volume of buffer TL proportionally. For
40mg of liver tissue, 400μL of buffer TL is required.

1A. For soft tissue, such as liver or brain, put the tissue into 1.5 mltube, add 200μL of Buffer TL, and homogenize thoroughly withmicrohomogenizer.
1B. If microhomogenizer is not available or the tissue is not soft,
grind the tissue to a fine powder with liquid nitrogen in a pre-cooledmortar and pestle. Put the powdered tissue into 1.5 ml tube. Add
200μL of Buffer TL and pulse-vortex for 15 seconds.
1C. If neither 1A nor 1B is available, mince the tissue with sharp
blade as small as possible. Put the tissue into a 1.5 ml tube. Add
200μL of Buffer TL and pulse-vortex for 15 seconds.
2. Add 20μL of Proteinase K solution. Mix by vortexing. Incubate at56℃ until the tissue is completely lysed.
If the sample is larger than 20 mg (if spleen, 10 mg), increase the amount ofProteinase K proportionally. For 40 mg of liver tissue, 40μL of Proteinase K
solution is required. Lysis time varies from 10 min to 3 hr usually depending on
the type of tissue processed and the lysis method used. The lysate will
become translucent without any particles after complete lysis. Overnight lysis
does not influence the preparation.
If the sample is lysed in water bath or heating block, vortex occasionally (2-3times per hour) during incubation to lysis readily. Lysis in shaking water bath,shaking incubator or agitator is best for efficient lysis.
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3. Check!! If Buffer TB precipitates, pre-heat in a 60℃ water bath todissolve completely.
4. Spin down the tube briefly to remove any drops from inside of thelid.
5. Optional: If RNA-free DNA is required, add 4μL of RNase solution(100mg/ml, not provided), vortex to mix thoroughly, and incubate
for 2 min at room temperature.
LaboPass TM Tissue spin column has the very stronger affinity to DNA thanRNA. Unless RNase is treated, RNA occupies very small portion of eluates.
RNA may inhibit some downstream enzymatic reactions, but will not inhibit
PCR.

6. Add 400μL of Buffer TB. Immediately vortex the tube to mix
thoroughly. Spin down the tube briefly to remove any drops frominside of the lid.
If the sample is larger than 20 mg (if spleen, 10 mg), increase the volume ofbuffer TB proportionally. For 40 mg of liver tissue, 800μL of buffer TB isrequired.

7. Apply the mixture to the spin column. Centrifuge for 1 min at
6,000 xg above(>8,000 rpm). Replace the collection tube with newone.(provided)
If more than 20 mg(10 mg spleen) of tissue is processed, apply the mixture
twice; apply 700μL of the mixture, spin down, discard the flow-through, reinsert
empty collection tube, and repeat the steps again until all of the mixture isapplied to the spin column. If the mixture has not passed completely throughthe membrane, centrifuge again at full speed until all of the solution has
passed through. Centrifuge at maximum speed will not affect the DNArecovery.

8. Add 700μL of Buffer BW. Centrifuge for 1 min at 6,000 xg
above(>8,000 rpm). Replace the collection tube with newone.(provided)
There is no need to increase the volume of Buffer BW and NW for larger
sample.

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9. Add 500μL of Buffer NW. Centrifuge for 3 min at 14,000 xg (fullspeed). Place the spin column in a clean 1.5 ml tube.(not provided)
Care must be taken at this step for eliminating the carryover of buffer NW. Ifcarryover of buffer NW occurs, centrifuge again for 1 min at full speed with theempty collection tube(not provided) before transferring to the new 1.5 ml tube.

10. Add 200μL of Buffer AE or distilled water. Incubate for 2 min atroom temperature. Centrifuge at 6,000 xg above(>8,000 rpm) for 1min.
Ensure that the buffer AE or distilled water is dispensed directly onto thecenter of spin column membrane for optimal elution of DNA. If higher
concentration of eluate is needed or starting amount of sample is very small,elution volume can be decreased to 50μL minimum. However, the small
volume of elution buffer will decrease the total amount of DNA recovery. For
long-term storage, eluting in buffer AE is recommended. But, EDTA included in
the buffer AE may inhibit subsequent enzymatic reactions, so you can avoid
such latent problems by using distilled water or Tris-Cl (>pH8.5). When using
water for elution, check the pH of water (>pH7.0) before elution.

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Protocols for Cultured Animal Cells
Before experiment- Prepare the water bath to 56℃- Equilibrate buffer AE to room temperature- Add appropriate volume of absolute ethanol to Buffer BW and NW before first use.
- All centrifugation should be performed at room temperature.
- Buffer TB may precipitate at cool ambient temperature. If so, dissolve it in 60℃
water bath.
- Mix Buffer TL and TB thoroughly by shaking before use.

Washing cells with sufficient volume of PBS before procedures usually brings
about better results.

1. Harvest cells (up to 5x106 cells) to a 1.5 ml microcentrifuge tube
by centrifugation at 14,000 xg for 10 sec.
Alternatively, cells can be pelleted at 500 xg for 5 min. For adherent cells,
trypsinize the cells before harvesting.

2. Discard the supernatant as much as possible and resuspend cellpellet in 200μL of Buffer TL.
3. Add 20μL of Proteinase K solution. Mix by vortexing. Incubate for10 min at 56℃.
Longer incubation will not affect DNA recovery.

4. Continue with Tissue Protocols from step 3.

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Protocols for Mammalian whole blood 300μL (Optional! Itneeds Buffer RL)
Before experiment- Prepare the water bath to 56℃- Equilibrate buffer AE to room temperature- Add appropriate volume of absolute ethanol to Buffer BW and NW before first use.
- All centrifugation should be performed at room temperature.
- Mix Buffer TL and TB thoroughly by shaking before use.
- Buffer TB may precipitate at cool ambient temperature. If so, dissolve it in 60℃water bath.

1. Transfer 300μL of whole blood to a new 1.5 ml tube.
Before transfer of blood, it is recommended that gently rocking the tube ofblood until throughly mixed.

2. Add 900μL of Buffer RL to the tube containing the blood sample.
Invert the tube 5-6 times to mix. Incubate the mixture for 10 min atroom temperature.
Invert 2-3 times during the incubation. The lysate should become translucent.
If the lysate is opaque not translucent, it may be frozen or mis-stored sample,
and you should read the annotation of step 3. If fresh or well-stored sample isprocessed, it will take less times than 10 min to acquire translucent mixtures.
This kit can process up to 1 ml of whole blood sample. If the sample is larger
than 300μL, see the detailed protocol for larger sample volume.

3. Centrifuge for 1 min at 14,000 xg (full speed) and carefully
remove the supernatant as much as possible without disturbing thevisible white (or pink) pellet. Resuspend the pellet by vortexing orflickering.
Approximately 10-20μL of residual liquid will remain. Steps 2-3 are criticalsteps for DNA recovery yields, so you have to check the translucent lysate andthe white (or pink) pellet before processing next steps. If blood sample has
been frozen or mis-stored, resuspend the pellet and repeat step 2-3 until
lysate become translucent.

4. Check!! If Buffer TB precipitates, pre-heat in a 60℃ water bath todissolve Completely.

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5. Add 200μL of Buffer TL to the tube containing the resuspended
cells. Pipet the solution 5-6 times to mix well.
The lysate may be viscous.

6. Add 10μL of Proteinase K solution. Mix by vortexing. Incubate for10 min at 56℃.
Overnight lysis is available and it will not influence the preparation.

7. Spin down the tube briefly to remove any drops from inside of thelid.
8. Optional: If RNA-free DNA is required, add 4μL of RNase solution(100 mg/ml, not provided), vortex to mix thoroughly, and incubate
for 2 min at room temperature.
LaboPass TM Tissue spin column has the very stronger affinity to DNA thanRNA. Unless RNase is treated, RNA will occupy very small portion of eluates.
RNA may inhibit some downstream enzymatic reactions, but will not inhibit
PCR.

9. Add 400μL of Buffer TB. Immediately vortex the tube to mix
thoroughly. Spin down the tube briefly to remove any drops frominside of the lid.
If Buffer TB precipitates, pre-heat in a 60℃ water bath to dissolve completely.

10. Apply the mixture to the spin column. Centrifuge for 1 min at
6,000 xg above (>8,000 rpm). Replace the collection tube with newone.(provided)
If the mixture has not passed completely through the membrane, centrifuge
again at full speed until all of the solution has passed through. Centrifuge atfull speed will not affect the DNA recovery.

11. Add 700μL of Buffer BW. Centrifuge for 1 min. at 6,000 xgabove(>8,000 rpm). Replace the collection tube with newone.(provided)

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12. Add 500μL of Buffer NW. Centrifuge for 3 min at 14,000 xg (fullspeed). Place the spin column in a clean 1.5ml tube.(not provided)
Care must be taken at this step for eliminating the carryover of buffer NW. Ifcarryover of buffer NW occurs, centrifuge again for 1 min at full speed with theempty collection tube(not provided) before transferring to the new 1.5 ml tube.

13. Add 200μL of Buffer AE or distilled water. Incubate for 2 min atroom temperature. Centrifuge at 6,000 xg above(>8,000 rpm).
Ensure that the buffer AE or distilled water is dispensed directly onto thecenter of spin column membrane for optimal elution of DNA. If higher
concentration of eluate is needed or starting volume of blood sample is very
low, elution volume can be decreased to 50μL minimum. However, the small
volume of elution buffer will decrease the total amount of DNA recovery. For
long-term storage, eluting in buffer AE is recommended. But, EDTA included in
the buffer AE may inhibit subsequent enzymatic reactions, so you can avoid
such latent problems by using distilled water or Tris-Cl (>pH8.5). When using
water for elution, check the pH of water (>pH7.0) before elution.

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Protocols for Mammalian whole blood 1 ml
(Optional! It needs Buffer RL.)
Before experiment- Prepare the water bath to 65℃- Equilibrate buffer AE to room temperature- Add appropriate volume of absolute ethanol to Buffer BW and NW before first use.
- All centrifugation should be performed at room temperature.
- Mix Buffer TL and TB thoroughly by shaking before use.
- Buffer TB may precipitate at cool ambient temperature. If so, dissolve it in 60℃
water bath.

1. Transfer up to 1 ml of whole blood to a new 15 ml tube.
Before transfer of blood, it is recommended that gently rocking the tube ofblood until throughly mixed.

2. Add 3 ml of Buffer RL to the tube containing the blood sample.
Invert the tube 5-6 times to mix. Incubate the mixture for 10 min atroom temperature.
Invert 2-3 times during the incubation. The lysate should become translucent.
If the lysate is opaque not translucent, it may be frozen or mis-stored sample,
and you should read the annotation of step 3. If fresh or well-stored sample isprocessed, it will take less times than 10 minutes to acquire translucent
mixtures.

3. Centrifuge for 10 min at 2,000 xg (3,000 rpm) and carefully
remove the supernatant as much as possible without disturbing thevisible white (or pink) pellet. Resuspend the pellet by vortexing orflickering.
Approximately 30-40μL of residual liquid will remain. Steps 2-3 are criticalsteps for DNA recovery yields, so you have to check the translucent lysate andthe white (or pink) pellet before processing next steps. If blood sample has
been frozen or mis-stored, resuspend the pellet and repeat step 2-3 until
lysate become translucent.

4. Check!! If Buffer TB precipitates, pre-heat in a 60℃ water bath todissolve completely.

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5. Add 200μL of Buffer TL to the tube containing the resuspended
cells. Pipet the solution 5-6 times to mix well. Transfer the lysate toa new 1.5 ml tube.
The lysate may be viscous.

6. Add 20μL of Proteinase K solution. Mix by vortexing. Incubate for10 min at 64℃.
7. Continue with Whole Blood 300μL protocols from step 7.

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