DNA Extraction of Animal tissue (Qiagen)

--- tags: molecular biology (tutorial) --- # DNA Extraction of Animal tissue (Qiagen) ##### *version 1.0: 2023-06-08* ## Summary This protocol is intended for the extraction of DNA from any animal sample containing tissue using the [Qiagen DNeasy Blood and Tissue Kit](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/dneasy-blood-and-tissue-kit). The protocol will yield sufficient quantities of DNA for a variety of downstream applications, including targeted PCR and Next Generation Sequencing (NGS) library preparation. This is a simplified and only slightly modified version of the manufacturer’s protocol. To get a broader understanding of the procedure, download and read the complete protocol from the [Qiagen website](https://www.qiagen.com/us/Resources/ResourceDetail?id=aa250d94-fc4b-4e27-bb74-d32391ff8a48&lang=en). :::info :bulb: Before Starting: 1. Check the extraction kit for sufficient supplies. If there is an insufficient number of collection tubes make new tubes by cutting tops off clean 1.5 mL tubes using scissors. You always need capless 1.5 mL tubes for the final elution step. One strategy is to fill a container with capless tubes ahead of time and autoclave it. 2. Clean and autoclave tubes and pestles. For cleaning used pestles, soak them in 10% bleach for 15 minutes, rinse well with DI/RO water, dry with clean lab wipes (kimwipes), and autoclave in a covered glass beaker. 3. Clean the lab bench by wiping down with 10% bleach, H2O, and 95% ethanol. Or use RNAse/DNA AWAY or similar. ::: ### Extraction Day 1: 1. Preheat a shaking incubator (heat block or equivalent) to 56°C. Check temperature with a thermometer and adjust if necessary. 2. Pull out and label the necessary number of 1.5 mL sample tubes. Mark tubes for non-destructive samples with “*ND*”. This is if you are using specimens that you want to deposit in a collection afterwards, or have vouchers for phylogenetic/taxonomic studies, etc. :::warning :warning: DO NOT GRIND “*ND*” SAMPLES! ::: 3. Pipet 180 μL of ATL buffer into each sample tube. Addition of ATL can be done before or after specimen is added to the tube, but I usually add the buffer first. 4. Carefully pull out and dry specimens and place them in the appropriate 1.5 mL sample tube containing buffer ATL (see extraction prep protocol for more detail). To avoid cross-contamination, it is a good idea to clean surfaces before starting and to sterilize your forceps between specimens (you can use a lab flamer or RNAse/DNA AWAY spray). 5. Carefully transfer sample tubes to the extraction lab (if not there already). 6. If destructive extraction, grind sample with pestle (you can use [Kimble tubes and pestles](https://www.dwk.com/na/kimble-disposable-pellet-pestle-tissue-grinder), they fit each other better) in buffer ATL until the specimen is completely fragmented. Carefully remove pestle from tube and check for small fragments. If needed, re-submerge the pestle in ATL buffer. Try not to remove specimen fragments or drops. Be careful to avoid getting liquid on your fingers or external tube surfaces. 7. Add 20 μL proteinase-K to each tube. Vortex each tube for 3-5 seconds and briefly spin to dislodge material from sides. 8. Place tubes in a 56°C shaking incubator overnight for lysis (8-14 hours - we suggest overnight lysis for ND samples). Overnight lysis might not be necessary for large, fresh specimens. Incubation for as little as 2 hours can be sufficient to lyse tissues. ### Extraction Day 2: 1. Make aliquots of each of the required reagents for the day: buffer AL, 100% EtOH, buffer AW1, buffer AW2, and buffer AE (or water). Multiply the number of samples by the amount of reagent needed for one sample and an error correction of ~1.1X. Making aliquots will keep the stock reagents from getting contaminated. Use the appropriate tube size. 2. Remove tubes from the incubator, vortex for 5 seconds and gently spin. *Optional:* Increase incubator temperature to 60°C and place buffer AE aliquot in incubator until elution step. Warming buffer AE is not necessary, but it can increase DNA yields during the elution step. 3. Add 200 μL buffer AL to each sample. Briefly vortex and spin. It is normal for a white precipitate to form. 4. Add 200 μL 100% EtOH to each sample. Briefly vortex and spin. 5. Pipet all liquid from each sample into a labeled DNeasy mini column, including any precipitate. If non-destructive extraction, leave the specimen in the tube. This can be done by either pushing the pipette tip to the bottom of the tube, or by gently pushing the specimen onto the side of the tube above the liquid. For destructive samples, try to avoid transferring specimen fragments to the column as these can clog the column and reduce yield. Fill the tube holding the ND specimen with 95% EtOH and set aside for later remounting. 6. Centrifuge mini columns for 1 min at 8000 rpm (6000 x g). Discard flow through and collection tube. Place DNeasy column in a new 2 mL collection tube. It is a good idea to visually inspect each column to make sure that all of the liquid flowed through the column during centrifugation. Centrifuge longer if necessary. 7. Add 500 μL buffer AW1. Repeat step 5 and 6. 8. Add 500 μL buffer AW2. Centrifuge for 3 min at full speed (14000 rpm). Discard flow-through and collection tube. 9. Place DNeasy columns in clean, capless 1.5 mL tubes (cut off caps with scissors). Do not use the Qiagen collection tubes; there are not enough. You could prepare a jar of capless tubes ahead of time. 10. Pipet 50-100 μL buffer AE (or water) into each sample and incubate at RT for 3 min. Centrifuge 1 min at 8000 rpm to elute DNA. The final elution volume can be varied from 100-200 μL, depending on size/age of specimen and desired DNA concentration. Also, nuclease-free water can be substituted for buffer AE. For insect matyerial, we generally do two rounds of 65 uL elutions for a final volume of 130 uL, incubating in RT for 3 min after each elution. Increase the volume if you think the specimen will yield plenty of DNA. 11. Repeat step 10 using the same elution volume. Do not discard the flow through and try not to let the eluate touch the column. 12. Transfer each eluate to a clean, labeled 0.5 mL tube. Immediately proceed to DNA QC or store the extracted stock DNA at -20°C for later use. We recommend labeling the tubes with the extraction number on the top and side. You can also print extraction codes for the top label so that it is easy to see and be read. ### DNA Quality Check (NGS Work): Use 2 μL of each sample to measure DNA concentration with [Qubit fluorometer](https://hackmd.io/20P-iQi6QH6B5BqfO6_Kww). Record values in your lab notebook and electronic extraction file. :::info **Optional**: Run 30-50 ng of gDNA on a gel. Avoid using more than 10 μL per sample. If the specimen was recently collected and properly preserved, this step may be skipped. Similarly, if the DNA concentration is very low, this step may be skipped to save DNA. An alternative method would be to run the sample on a Tape Station or BioAnalyzer instrument. ::: ### ND Specimen Clean-up 1. Following extraction, non-destructively-sampled specimens should be placed immediately into a tube of 95% EtOH. After anywhere from 10 minutes to a day later, specimens should be dried and re-mounted.