IBI DNA/RNA/Protein Extraction Kit - 50 Preps
- Sample Size: up to 5×106 cultured animal cells/up to 25mg animal tissue /up to 500µl whole blood/up to 200µl biological fluids (serum, plasma)
- Expectant Yield: up to 9µg of genomic DNA/20µg of total RNA/120µg of protein from 1×106 of HeLa cells
- Operation Time: DNA/RNA purification within 25 minutes, protein precipitation within 50 minutes
- Elution Volume: 50 – 200µl (genomic DNA)/25 – 50µl (total RNA)
- Format: genomic DNA spin column and total RNA spin column
The DNA RNA Protein isolation Kit provides an efficient method for purifying genomic DNA, total RNA, and total protein simultaneously from cultured cells, animal tissues, whole blood, and biological fluids. Chaotropic salt is used to lyse cells and inactivate DNases and RNases, allowing DNA to bind to the genomic DNA spin column. The flow-through can then be transferred to the RNA Spin column for RNA binding. Contaminants are effectively removed using wash buffers followed by pure genomic DNA elution in a low salt buffer and pure total RNA elution in RNase-free water.
DNA/RNA purification can be completed in 25 minutes without phenol/chloroform extraction or alcohol precipitation, and protein purification can be completed in 50 minutes. The purified DNA, with approximately 20 – 30Kb, is suitable for use in PCR or other enzymatic reactions and the purified RNA (including miRNA) is ready for use in RT-PCR, Real-Time PCR, northern blotting, primer extension, mRNA selection, and cDNA synthesis. The purified proteins can be directly analyzed on a SDS-PAGE and subsequent western blot.
The quality of the DNA RNA Protein isolation Kit is tested on a lot-to-lot basis by isolating genomic DNA and total RNA from 1x106 cultured animal cells. The purified DNA and total RNA is quantified with a spectrophotometer and analyzed by electrophoresis on a 1% agarose gel. The purified protein is quantified by Bradford assay analyzed on SDS-PAGE.
Sample: Cultured cells, animal tissue, whole blood & biological fluids
Format: Genomic DNA spin column and Total RNA spin column.
Yield: up to 9ug of Genomic DNA / 20ug of Total RNA / 120ug of Protein from 1 x 106 HeLa cells.
Operation Time: Within 25 minutes
Elution volume: 50 – 200ul for Genomic DNA / 25 – 50ul for Total RNA.
Simultaneous extraction of Genomic DNA and Total RNA from cultured cells, animal tissues, whole blood and biological fluids.
Is the protein denatured? Yes, the DR Buffer used in the lysis step contains strong denaturing components. Thus, the proteins will denature during the lysis step. One purpose of the DR Buffer is to inactivate all RNAse and proteases.
Fragment sizes of DNA & RNA that can be extracted? 1.The average size of genomic DNA is between 20-30 kb. 2.All RNA fragments larger than 200 NT can be purified. However, some small RNAs and TRNAs (> 100 NT) can also be collected from the column.
Can I use frozen cells with this kit? Yes, after removing cell sample from freezer, add DR buffer (premixed with B-ME) to the cell pellet immediately, and re-suspend by pipetting.
Can I use the extracted protein on a Native gel? No, because the protein was denatured in the lysis step.
Are the mitochondria DNA present in the extracted Total DNA? Theoretically, the mitochondria DNA should be present in the Total Extracted DNA however; we have NOT tested for this to be certain.
I am having issues re-suspending the protein pellet, any suggestions? Change the pH of the suspension buffer. Use a different suspension buffer using a different detergent. Try using 5% SDS to dissolve the pellet.
Can I use a lysis/RIPPA buffer to dissolve the protein pellet after? What is the DV buffer? You can use a RIPPA buffer to dissolve the protein pellet. However, if the pellet is difficult to dissolve the DV buffer or a 5% SDS solution may work better. Once the pellet is dissolved, mix the protein with 2 X Laemelli buffer and load on to your gel.
Can this kit be used on samples for ChiP protocol? Probably not, since most samples for that protocol have been treated with formaldehyde.
Can the kit be used on paraffin embedded samples? Treat the sample with xylene and wash with absolute ethanol, then proceed to use the kit.
Will this kit pull down membrane proteins? Since the DR buffer contains Triton X-100 you should be able to precipitate the protein using acetone.
Will the DNA-RNA-Protein kit work on brain tissue samples? This kit is optimized for use on easy-to-lyse samples. Brain tissue has a high fat content and will not work well in this kit. Recommend the Tri-Isolate kit instead.
Why am I having low A260 /230 ratios? The low 260/230 most likely resulted from salt contamination in the DNA product. Wash the GD column twice to remove salt on the membrane prior to DNA elution.