Protocol for DNA extraction by bead mill and SDS lysis May 29, 2013

Transcription

1 Protocol for DNA extraction by bead mill and SDS lysis May 29, 2013 Before using this protocol, test your samples by extracting DNA from a typical soil in your soil sample set using the MO BIO PowerSoil DNA Isolation Kit (MO BIO, ). Check DNA shearing with an agrose gel (0.8%, 105 V, 30 min). If DNA shearing is negligible, continue using the PowerSoil kit for all these type of your soil samples. If DNA shearing is visible but not very serious, you can try this protocol to reduce DNA shearing by combining SDS lysis and using our classic Tris buffer (details below). You can also try different soil load weights and bead time to determine the optimum conditions for your samples. If DNA shearing is serious, you should use the classic protocol with grinding and SDS lysis followed by gel purification ( Community DNA Preparation through hydridization at * This modified method does not improve DNA purity or apparent DNA yield compared to using the recommended PowerSoil kit protocol (DNA yield 75%~110% of recommended PowerSoil kit protocol). ** Some steps in this protocol are the same as in the recommended protocol of the PowerSoil DNA isolation kit. You may check the protocol of PowerSoil to understand the steps better ( Materials For 24 samples Tris buffer (ph=8.0, this is the same buffer as our classic protocol on the IEG website): 40 ml Extraction buffer 6.8 ml 1 M NaH 2 PO 4 (monobasic) 93.2 ml 1 M Na 2 HPO 4 (dibasic) Combine phosphate solutions, adjust ph to 8.0 with NaOH, continue with remaining ingredients 200 ml 0.5 M EDTA, ph ml 1 M Tris-HCl 300 ml 5 M NaCl 100 ml 10% CTAB (Final concentrations: 0.1 M NaH 2 PO 4, 0.1 M Na 2 HPO 4, 0.1 M EDTA, 0.1 M Tris-HCl, 1.5 M NaCl, 1% CTAB) Bring to 1 L with DI water, if CTAB is left out; bring to 900 ml with DI water (Final concentrations: 0.1 M NaH 2 PO 4, 0.1 M Na 2 HPO 4, 0.1 M EDTA, 0.1 M Tris-HCl, 1.5 M NaCl, 1% CTAB) 1 / 5

2 SDS (20%): 4.5 ml Isopropanol: 20 ml Ethanol (70%, ice cold): 50 ml Solutions and tubes from PowerSoil Kit (C1 and C6 are not needed for this protocol) Beadtube (with bead and bead solution): 24 preps (1/sample) Solution C2: 6.2 ml (250 µl/sample) Solution C3: 5 ml (200 µl/sample) Solution C4: 30 ml (1.2 ml/sample) Solution C5: 12.5~25 ml (0.5~1 ml/sample) Spin Filter (units in 2 ml tubes): 24 preps (1/sample) 2 ml collection tube: 24 tubes (1/sample) 1.5 ml tube: 24~48 tubes (1~2/sample, can also use 2 ml tubes) 2 ml tube: 48 tubes (2/sample) 15 ml tube: 1 (to keep bead solution) 1 ml tip: ~200 tips 200 µl tip: ~30 tips 10 µl tips: 30~80 tips 5 ml tip: 1 (for solution C4) Vortex (with 24-place MO BIO Vortex Adapters for Vortex-Genie 2): 1. Water bath (65ºC, 37ºC, 50ºC), timer, spoons, centrifuge and other common materials. Steps and notes 1. Bead mill (~1.5 h for 24 samples) (1) Transfer the solution (600~650 µl) from the bead tube in the MO BIO PowerSoil Kit to a clean tube. There should only be beads remaining in the bead tube. Note: Do not discard the bead solution, you will need it at step (13). (2) Weight 0.5 g soil into the bead tube. Note: The best loading weight may vary for different soils. Larger amounts of soil may significantly increase DNA shearing. If you need more DNA but can not accept any more DNA shearing, extract 2 aliquots of 0.5 g soil (1 g total) separately. (3) Add 900 µl Tris buffer and 100 µl 20% SDS to bead tube. (4) Vortex the bead tube for 10 min at maximum speed. Note: If DNA yield is too low and the shearing is not significant, you can try vortexing for a longer amount of time. If the sample lyses easily, you can use a shorter time to decrease DNA shearing and could use the PowerSoil kit protocol. * Attention: the number of bead tubes in the Vortex Adapter has an obvious effect on lysis 2 / 5

3 efficiency, as mentioned in the MO BIO PowerSoil kit protocol.so you had better use the same number of bead tubes on the Vortex Adapter every time (sometimes you can use some fake tubes). Otherwise, you need to change the vortex time to get the same lysis efficiency. ** Attention: Bead time is critical to limit DNA shearing. The best bead time for one soil may not be the same for another soil. A 20-min vortex may cause serious DNA shearing for some soil samples. Please test vortexing times for your sample to get optimum bead time before doing large amount samples. 2. SDS incubation (~1 h busy time and 1 h free time for 24 samples) (5) Incubate the bead tube at 65 for 1 h. Note: 1 hour is recommended to get more intact DNA, although DNA yield rates may decrease a bit. If DNA shearing is still too much, try a shorter incubation time. * Too long an incubation will cause serious DNA shearing. ** If you do not mind a bit more time cost, you can transfer all the contain of a bead tube to a 15-ml tube and add more buffer (another 900ul Tris buffer and 100ul 20% SDS) before incubation at 65C. You may get higher DNA yield and even less DNA shearing. (6) Centrifuge at 10,000 x g for 5 min. Transfer supernatant to a clean 2 ml tube. Note: Try your best to avoid the white layer.the volume of transferred supernatant may be 600~650 µl (sometime may be more). (7) Add 720 µl Tris buffer and 80 µl 20% SDS to precipitant. Mix well. Incubate at 65 for 15 min. Centrifuge at 10,000 x g for 5 min. Transfer supernatant to the 2 ml tube to combine with supernatant from the previous step. Note: Try your best to avoid the white layer. The volume of transferred supernatant may be 600 µl. You can use 900µl Tris buffer and 100µl 20% SDS at this step and the DNA yield will be more, but you will need a bigger tube in next step. * If skipping this step, the DNA yield may decrease by 30%~35%. 3. Isopropanol precipitation (~1 h busy time and 1 h free time for 24 samples, excluding overnight precipitation) (8) Add 0.6 volume isopropanol and store at -20 or -80 overnight. Note: If following the above volumes, 720 µl isopropanol will be added to 1.2 ml sample (1.2 x 0.6 = 720). If the volume will exceed 2 ml after adding isopropanol, you can divide the sample into 2 tubes before adding isopropanol or just use a bigger tube. It is important to use 0.6 volumes as exactly as possible. * If using ammonium acetate for precipitation, DNA yield may decrease by ~27%. 3 / 5

4 (9) Warm at 37 to dissolve any mineral precipitates. Note: usually 20~30 min. (10) Centrifuge at 15,000g for 20 min. (11) Discard supernatant. Wash the pellet with 1 ml ice-cold 70% ethanol twice. Discard ethanol. Note: There may be some isopropanol remaining in the lid after the first washing. To avoid this vortex briefly when washing the pellet to make sure the ethanol washes the entire tube and then centrifuge to reset the pellet. Otherwise, you can transfer the pellet to a clean tube when washing. Discard as much ethanol as possible, and then you do not need to dry the pellet. If you divided a sample to 2 tubes at step (8), you can combine the pellets into 1 tube at this step before washing. (12) Dissolve the pellet in 30 µl nuclease free water. Incubate at 50 for 5 min if the pellet is hard to dissolve. Make sure the pellet is dissolved well before proceeding to the next step. Note: If you need to know the DNA concentration in a crude DNA sample, save 2~3 µl crude DNA sample and do Picogreen test as soon as possible ( dsdna quantification with PicoGreen at Crude DNA will have contaminants which may cause DNA degradation especially when dissolved in water (even when stored at -20ºC), thus it is better to do quantification as soon as possible. 4. DNA purification (~4~5 h busy time for 24 samples, not including step (24)) (13) Add 430 µl bead solution (MO BIO PowerSoil kit) to dissolved DNA. Mix to make sure DNA is dissolved well. Note: You can use desktop vortex (1 min at the minimum speed should be enough). Solution C1 in the PowerSoil kit is an SDS solution which is not needed in this protocol. (14) Add 250 µl solution C2, vortex 5 s to mix, incubate at 4 for 5 min. (15) Centrifuge at 10,000g for 1 min. Transfer 650 µl supernatant to a clean tube. (16) Add 200 µl solution C3, vortex 5 s to mix, incubate at 4 for 5 min. (17) Centrifuge at 10,000g for 1 min. Transfer 700~750 µl supernatant to a 2 ml tube. (18) Add 1.2 ml solution C4, vortex 5 s to mix. 4 / 5

5 (19) Load 675 µl of the sample to the Spin filter (the column). Centrifuge at 10,000 g for 1 min. Discard the flow through. Repeat twice using the same Spin filter for a total of 3 times. (20) Add 500 µl solution C5 to the Spin filter and centrifuge at 10,000g for 30 s. Discard the flow through. Repeat if your sample is too dirty. Note: it is not necessary to wash more than 2 times with C5. The 260/230 ratio may not be better. * In my test, washing twice or more is the same as washing once. (21) Centrifuge Spin filter one more time at 10,000g for 1 min. Carefully transfer the Spin filter to a new 2 ml tube. Note: Be careful. Some solution C5 may remain on the rim (you can use a clean kimwipe to blot it). (22) Add 100 µl nuclease free water to the center of the filter. Centrifuge at 10,000g for 30 s. Discard the Spin filter. Note: Warmed water and eluting twice has little effect on result. Incubating the spin filter at 50 can significantly decrease DNA yield. (23) Check DNA purity with Nanodrop. 260/280 ~ 1.8, 260/ Note: If 260/230 is bad, do desalting according to Community DNA Preparation through hybridization at (page 10, Desalting protocol). If the DNA concentration is too low, it will be hard to precipitate DNA by acidification and cold ethanol. You may concentrate the DNA to about 100 ng/μl, precipitate DNA and wash the pellet with 10~20 volumes 70% cold ethanol. (24) Determine the dsdna concentration by Picogreen test ( dsdna quantification with PicoGreen at Check DNA shearing with agrose gel (0.8% agrose, 105 V, 30 min). Note: You can optimize the soil load weight at step(2) and vortex bead beating time in step(4) according to DNA quality and quantity. Please feel free to contact Daliang (ningdaliang@gmail.com) for any question or suggestion. 5 / 5