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Lolina A/S

Address: Sindalsvej 30 8240 Risskov Danmark 

                    Email: Info@lolina.dk

 Website: https://lolina.dk

Product Specification

Product Description

The Lolina?  Micro DNA Kit utilizes Lolina?  DNA Column M3 and a novel  solution system,  suitable for rapid extraction of genomic DNA from trace  samples such as blood, forensic materials, dried blood spots, prescription pads,  chewing gum, urine,  and other trace samples. The extraction process does not require  the  use  of toxic  organic  compounds  such  as  phenol-chloroform  or  time-consuming  ethanol precipitation. It  is  easy to  operate  and  can  complete the DNA extraction  and purification  of a  single sample within 30 minutes. The Lolina?  DNA Column M3  in the kit selectively adsorbs nucleic  acids without  adsorbing proteins, polysaccharides,  and  other non-nucleic  acid  substances, resulting  in high DNA purity suitable for direct use in PCR, qPCR, and other experiments.

Components

Transportation and Storage Methods

Precautions:

1.   Avoid prolonged exposure of reagents to air to prevent evaporation, oxidation, and changes in pH. Always close the lids tightly  after using each solution.

2.    Precipitation may occur when the Binding Buffer (BD) and Protein Lysis Buffer (PL) are  stored at low temperatures. If this happens, gently heat the  solutions at 37°C until they become clear again. This does not affect their effectiveness.

3.    The Binding Buffer  (BD) and Protein Lysis Buffer (PL) contain irritating compounds. Wear latex  gloves during operation to prevent  skin contact. In case of skin or eye contact, rinse with plenty of water or saline solution immediately.

    4.    For your safety and health, wear a lab coat and disposable gloves while handling the reagents.

    5.    This product is intended for research purposes only.

Pre-experiment Preparation:

1.    Prepare your  own equipment and reagents: Benchtop centrifuge, shaker, water bath or metal bath, 100% ethanol, liquid nitrogen (for tissue grinding),  1.5 mL centrifuge tubes,  etc.

2.    All  centrifugation  steps  should be performed at room temperature using a conventional benchtop centrifuge capable of reaching  12 , 000 rpm.

3.    Before the first use, add four times the volume of anhydrous ethanol to the wash buffer (W*) bottle, mix  thoroughly,  and   label  it.   If there   is  a  significant  discrepancy  in  volume   due  to  improper  transportation or storage, use a graduated cylinder to adjust the volume  and then add the specified  volume  of anhydrous ethanol. After each use, tightly  close the bottle cap to maintain the  ethanol

content.

4.    Carrier RNA usage: If the  starting material is limited, it is recommended to use Carrier RNA. If a  large  amount  of nucleic  acid  yield  is  expected,  users  can  choose  whether  to  add  Carrier  RNA  according to their needs. When using,  add 4  μL of Carrier RNA  storage  solution to the Binding  Buffer  (BD) required  for  each  sample  extraction,  and mix  the Binding  Buffer  (BD)  and  Carrier  RNA solution thoroughly by inversion  (Binding Buffer (BD) tends to foam,  so do not use vortex  mixing). Alternatively, you  can add the total amount of Carrier RNA needed to the total amount of Binding Buffer (BD) required and mix well for later use. The mixture is stable at room temperature  for up to 24 hours.

Operating Procedure:

1)      Pre-processing of Samples:

l Blood Sample

1.   Pipette  10-100  μL of blood into a  1.5 mL centrifuge tube. Add  10  μL of Proteinase K (20 mg/mL) and vortex thoroughly immediately to ensure thorough mixing.

Note: If less than  100  μL of blood is available, top up with lysis buffer LB.

    2.    Add  100 μL of Binding Buffer (BD) and mix thoroughly. Incubate at 70 ° C for  10 min.

Note:  If low  DNA  yield  is  expected,  it  is  recommended  to  add   1  μL  of Carrier  RNA  storage solution to  100  μL of Binding Buffer (BD) .

3 .   After   cooling,   add  50   μL  of anhydrous  ethanol  (pre-cooled)  and  vortex  immediately  to  ensure thorough mixing. Briefly centrifuge to collect droplets from the inside of the tube cap.

    4.     Allow to stand at room temperature (15-25 ° C)  for 3 min.

l Blood Card Sample

1.   Use  a  hole  puncher  to  collect  2-3  pieces  of 3  mm  diameter  blood  card  paper  into  a   1.5   mL centrifuge tube. Add  180  μL of lysis buffer LB and 20  μL of Proteinase K (20 mg/mL) , and vortex immediately to ensure thorough mixing.

    2.    Place the tube on a 56 ° C orbital shaker at 900 rpm for  1 hour.

Note: Alternatively, the tube  can be placed  in  a  56°C water bath  or metal bath  for  1 hour, with vortexing for  10  seconds every  10  minutes during incubation.

    3.   Add 200 μL of Binding Buffer (BD) and mix thoroughly.

    4.    Place the tube on a 70 ° C orbital shaker at 900 rpm for  10 minutes.

Note: Alternatively, the tube can be placed in a 70°C water bath or metal bath for  10 minutes, with vortexing for  10 seconds every 3 minutes during incubation.

l Microscopic Tissue

     1.   Collect  a  small  amount  of tissue  (<   10  mg)  into  a   1.5  mL  centrifuge  tube. Add  180  μL of lysis buffer  LB  and  20  μL  of Proteinase  K  (20 mg/mL),  and  vortex  immediately  to  ensure  thorough mixing.

      2 .   Incubate the tube in  a 56 ° C water bath or metal bath for   1  hour until the  solution becomes clear, gently shaking a few times during incubation to aid in lysis.

    3.   Add 200 μL of Binding Buffer (BD) and mix thoroughly.

Note:  If low  DNA  yield  is  expected,  it  is  recommended  to  add  2  μL  of Carrier  RNA  storage solution to 200  μL of Binding Buffer (BD) .

    4 .   After  cooling,   add  200  μL  of anhydrous  ethanol  (pre-cooled)  and  vortex  immediately  to  ensure

thorough mixing. Briefly centrifuge to collect droplets from the inside of the tube cap.

    5.   Allow to stand at room temperature (15-25 ° C)  for 5 minutes.

l Chewing Gum

     1.   Collect  30 mg of chewing gum into a   1.5 mL centrifuge tube. Add 280  μL of lysis buffer LB and 20  μL of Proteinase K (20 mg/mL) ,  and vortex immediately to ensure thorough mixing.

    2.    Place the tube on a 56 ° C orbital shaker at 900 rpm for 3 hours.

Note: Alternatively, the tube  can be placed in a  56°C water bath  or metal bath  for  3 hours, with vortexing for  10  seconds every  10  minutes during incubation.

   3.   Add 200 μL of Binding Buffer (BD) and mix thoroughly.

Note:  If low  DNA  yield  is  expected,  it  is  recommended  to  add  2  μL  of Carrier  RNA  storage solution to 200  μL of Binding Buffer (BD) .

    4.    Place the tube on a 70 ° C orbital shaker at 900 rpm for  1 hour.

Note: Alternatively, the tube  can be placed  in  a  70°C water bath  or metal bath  for  1 hour, with vortexing for  10  seconds every  10  minutes during incubation.

5 .  After  cooling,   add  200  μL  of anhydrous  ethanol  (pre-cooled)  and  vortex  immediately  to  ensure thorough mixing. Briefly centrifuge to collect droplets from the inside of the tube cap.

    6.   Centrifuge at  12 ,000 rpm for 5 minutes and collect the supernatant.

l Forensic Samples

   1.    Cigarette Butt:

Take a  1  cm2 piece of cigarette butt and cut it into 6 pieces. Transfer them to a  1.5 mL centrifuge tube.  Add  300   μL  of  lysis  buffer  LB   and  20   μL  of  Proteinase  K   (20  mg/mL),   and  vortex immediately to ensure thorough mixing.

Hair:

Cut 0 . 5-1  cm of hair close to the hair follicle and transfer it to a  1 . 5  mL centrifuge tube. Add 280 μL  of lysis  buffer  LB,  20   μL  of Proteinase  K  (20  mg/mL) ,  and  20   μL  of  1  M  DTT  solution (self-prepared), and vortex immediately to ensure thorough mixing.

    2.    Place the tubes on a 56 ° C orbital shaker at 900 rpm for  1 hour.

Note: Alternatively, the tubes can be placed in a  56°C water bath  or metal bath  for  1 hour, with vortexing for  10  seconds every  10  minutes during incubation.

   3.    Add 200 μL of  Binding Buffer (BD) and mix thoroughly.

Note:  If low  DNA  yield  is  expected,  it  is  recommended  to  add  2  μL  of Carrier  RNA  storage solution to 200  μL of Binding Buffer (BD) .

   4.   Place the tubes on a 70 ° C orbital shaker at 900 rpm for  1 hour.

Note: Alternatively, the tubes  can be placed in a 70°C water bath  or metal bath  for  1 hour, with vortexing for  10  seconds every  10  minutes during incubation.

   5.   Centrifuge at  12 ,000 rpm for 5 minutes and collect the supernatant.

2)   DNA Extraction:

    1 .   Insert the DNA binding column M3  into a 2 mL collection tube and set aside.

2 .   Add  the pre-processed   sample  solution  into the  DNA binding column M3 .  Centrifuge  at  12 , 000 rpm for  1 minute and discard the flow-through.

Note: The maximum volume  of the mixed  solution  added  each time  should not  exceed  650  μL. Multiple centrifugation steps may be necessary.

3.    Add 500  μL of Proteinase K buffer (PL) to the column.  Centrifuge at   12 ,000 rpm for 30  seconds and discard the flow-through.

4.   Add  600  μL of wash buffer to the column.  Centrifuge at   12 ,000 rpm at room temperature for 30 seconds and discard the  flow-through.

Note: Ensure that the wash buffer contains anhydrous ethanol.

   5.    Repeat step 4 once.

6 .   Place the DNA binding column M3  back into the  collection tube  and centrifuge at   12 , 000  rpm at room temperature for 2 minutes to remove any residual wash buffer.

7 .   Place the DNA binding  column M3  into a new   1. 5  mL centrifuge tube. Add 20-50  μL of elution buffer to the  center of the column . Let it stand at room temperature  for 2 minutes. Then centrifuge at  12 ,000 rpm for  1 minute. Collect the filtrate, which is the DNA solution.

Note: To increase the yield, the  elution buffer can be preheated to 70°C. Additionally, for higher yields, the DNA filtrate can be applied to the column again, left at room temperature for 2 minutes, and then eluted.

    8.   The DNA solution can be stored long-term at -20 ° C.

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