TITLE: Qiagen All-Prep DNA/RNA Mini Kit
AUTHORS: George  Testo

[DESCRIPTION]
The AllPrep DNA/RNA Mini Kit is designed to purify genomic DNA and total RNA simultaneously from a single biological sample. Lysate is first passed through an AllPrep DNA spin column to selectively isolate DNA and then through an RNeasy spin column to selectively isolate RNA. Pure DNA and RNA are purified from the entire sample, in contrast to other procedures where either the biological sample or the purified total nucleic acids is divided into two before being processed separately. The kit is compatible with small amounts of a wide range of animal cells and tissues.

The AllPrep DNA/RNA Mini Kit allows the parallel processing of multiple samples in less than 40 minutes. Time-consuming and tedious methods, such as CsCl step-gradient ultracentrifugation and alcohol precipitation steps, or methods involving the use of toxic substances, such as phenol and/or chloroform, are replaced by the AllPrep DNA/RNA procedure.

Genomic DNA purified with the AllPrep DNA/RNA procedure has an average length of 15–30 kb depending on homogenization conditions. DNA of this length is particularly suitable for PCR, where complete denaturation of the template is important to achieve the highest amplification efficiency. The purified DNA is ready to use in any downstream application, including:
Next-generation sequencing (NGS)
PCR
Southern, dot and slot blot analyses
Comparative genome hybridization (CGH)
Genotyping, SNP analysis

[STEPS]
SECTION: Homogenization
1. Add 600 µL of   to Lysing Matrix E tubes.

SECTION: Homogenization
2. Place the entire tissue or a portion into Lysing Matrix E tubes for homogenization.
SECTION: Homogenization
2.1. For Swab(s): pick up with tweezers and cut with surgical scissors sterilized in a Germinator 500. Place the cut end of a single swab into the Lysing Matrix E tube.
SECTION: Homogenization
2.2. 
For Nasal Lavage(s): Thoroughly thaw the lavage and pipette up and down 6 times. Then transfer 500 µL of the lavage to the Lysing Matrix E tube.

SECTION: Homogenization
3. Homogenize the lysate in Buffer RLT Plus (do not use more than 30 mg of tissue).
SECTION: Homogenization
3.1. Use a homogenizer designed for Lysing Matrix E tubes.
SECTION: Homogenization
3.2. Use a disruption setting that works best for your tissue (i.e. fecal, lung, etc).
SECTION: Homogenization
4. 
Centrifuge the lysate for 3 min at maximum speed. Carefully remove supernatant by pipetting and transfer it to the All-Prep DNA spin column placed in a 2mL collection tube.

SECTION: Homogenization
4.1. Close the lid gently, and centrifuge for 30 s at 10.000 rpm.

 

SECTION: Homogenization
5. 
Place the All-Prep DNA spin column in a new 2mL collection tube, and store at room temperature (15-25C) or a 4 °C for later DNA purification in the following steps.

 

SECTION: Total RNA Purification
6. Add 1 volume (~1x) of 70% Ethanol to the flowthrough from step 5 and mix well by pipetting. Do not centrifuge. Proceed immediately to step 7.

If some lysate was lost during homogenization, adjust the volume of ethanol.

 

SECTION: Total RNA Purification
7. Transfer up to 700 µL of the sample, including any precipitate that may have formed, to a RNeasy spin column placed in a 2mL collection tube. Close the lid gently, and centrifuge for 15 s at 10.000 rpm. Discard the flowthrough.

SECTION: Total RNA Purification
7.1. Reuse the collection tube in step 7.

 

SECTION: Total RNA Purification
8. Add 700 µL of  to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 10.000 rpm to wash the spin column membrane. Discard the flow-through.

SECTION: Total RNA Purification
9. Add 500 µL of   to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 10.000 rpm to wash the spin column membrane. Discard the flow-through.

SECTION: Total RNA Purification
9.1. Reuse the collection tube in step 9.

 

SECTION: Total RNA Purification
10. 
Add 500 µL of   to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at 10.000 rpm to wash the spin column membrane.

 

 


SECTION: Total RNA Purification
11. Place the RNeasy spin column in a new 2mL collection tube and discard the old collection tube with the flowthrough. Centrifuge at full speed for 1 min.

 


SECTION: Total RNA Purification
12. Place the RNeasy spin column in a new 1.5ml collection tube. Add 30-50 µL of   directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 10.000 rpm to elute the RNA.

SECTION: Total RNA Purification
12.1. If expected RNA yield is >30ug, repeat step 12 using another 30-50 µL of , or using the eluate from step 12 (if high RNA concentration is required).

SECTION: Genomic DNA Purification
13. Add 500 µL of   to the All-Prep DNA spin column from step 5. Close the lid gently, and centrifuge for 15 s at 10.000 rpm. Discard the flow-through.

SECTION: Genomic DNA Purification
13.1. Reuse the spin column in step 13.

 

SECTION: Genomic DNA Purification
14. Add 500 µL of   to the All-Prep DNA spin column. Close the lid gently, and centrifuge for 2 min at full speed to wash the spin column membrane.

 



 


 

SECTION: Genomic DNA Purification
15. Place the All-Prep DNA spin column in a new 1.5mL collection tube. Add 50 µL of   directly to the spin column membrane and close the lid. Incubate at room temperature (15-25 °C) for 1 min, and then centrifuge for 1 min at 10.000 rpm to elute the DNA.


SECTION: Genomic DNA Purification
16. Repeat step 15 to elute further DNA.