Streptococcus DNA Extraction
- 50mM Tris-10mM EDTA, pH 8 Autoclaved.
- Note: max concentration that will dissolve EDTA (without altering pH) is 0.1M. Making 10mL of the 50mM Tris Buffer Solution + 10mM of the EDTA will result in a 10% diluted Tris Buffer solution. We are working to troubleshoot this problem for the next time around.
- If working with small volumes such as 10mL that need to be sterilized, an alternative to autoclaving is filter sterilization. Use either a 10 or 20mL syringe and 0.2 micro filter tip.
- 10 mg/ml lysozyme
- 10 mg/ml protease K
- XXmg/ml RNAase A
- 10% SDS
- Tryptic soy broth blood plates
- Tryptic soy broth
Growing Up Strains
- From glycerol stocks in -20°C freezer, get the strains you want to work with.
- Hermans 930
- Pmen 4
- Day 1:
- Get Blood Plates (1 per strain), inoculation loops, tube holder. Bring everything into the hood.
- Using an inoculation loop, streak for single colonies onto the entire plate. Obviously change loops between strains.
- Incubate plates upside down, O/N at 37°C
- Day 2:
- Using a sterile cotton swab, pick single colonies and streak them over a half tryptic soy broth blood plate.
- Incubate O/N at 37°C
- Day 3:
- For each strain, prepare 2 microcentrifuge tubes of 1ml tryptic soy broth each. Using a sterile cotton swab, transfer half of a blood plate of growth into a microcentrifuge tube with 1ml tryptic soy broth. Using the same cotton swab, swab this same half of the plate to get all bacteria off of it.
- The salt is needed to precipitate the DNA in ethanol in later stages.
- START extracting genomic DNA no later than 6 hours after you place cells in broth.
- Ideally, start right away.
- Turn on small water bath, set for 37°C.
- Spin down one tube per strain at full speed for 1 minute.
- Remove supernatant. Just shake out the liquid, no need to pipette it out.
- Pipet in 1ml of cells from the other TSB tube for the strain.
- Remove as much medium as possible with a pipette, but do not remove any cells.
- Add 450uL of 50mM Tris-10mM EDTA, pH 8. Vortex to resuspend cells.
- If having trouble resuspending cells, then let sit for 10-15 minutes before trying again.
- Add 50uL 10 mg/ml lysozyme.
- Incubate in 37°C water bath for 10 minutes.
- Add 5uL 10 mg/ml protease K. Vortex to mix.
- Incubate in 37°C water bath for 30 minutes.
- Add 5uL XXmg/ml RNAase A and 50uL of 10% SDS
- Invert tubes several times to mix.
- Incubate in 37°C until the cultures are clear, this may take anywhere from 10 minutes to an hour.
- Occasionally invert them to mix.
- Spin them down at max speed for 10 minutes.
- Remove the top 500uL (ONLY this volume) supernatant without disturbing the cell pellet.
- Transfer this volume to a new micro-centrifuge tube.
- If you disturb the pellet, re-spin. This is really important, don’t transfer any of the pellet.
Genomic Extraction of DNA
Use the DNA extraction kit (Zymogen Genomic DNA Concentration and Extraction Kit)
- Turn on the dry water bath and incubate a microcentrifuge tube of the DNA Elution Buffer or water (pH > 6.0) at 65°C
- Add EtOH to the Binding Buffer as specified on the label. If you added EtOH, be sure to label such on the top cap and on the label. Then, be sure to close this bottle tightly each time you use it.
- Obtain one 1.5mL microcentrifuge tubes per strain and transfer 250uL of the lysed cell solution, so that there are two 1.5mL microcentrifuge tubes with 250ul each of lysed cells. Add 500uL of ChIP DNA Binding Buffer to each tube. Mix thoroughly through gentle inversion (not vortexing).
- Do not be tempted to put this in one microcentrifuge tube; the contents will be at capacity and the lysed cell solution will splash out, potentially contaminating your gloves and/or the other DNA solutions.
- Transfer one tube of this mixture to the Zymo-Spin IC-XL Column in a Collection Tube
- Centrifuge (between 10,000-16,000 x g) for 30 seconds. Discard the flow through.
- Transfer the other tube of this mixture to same column.
- Centrifuge (between 10,000-16,000 x g) for 30 seconds. Discard the flow through.
- Add 200uL DNA Wash Buffer to the column. Centrifuge for 1 minute. Repeat this wash step.
- Add at 20uL DNA Elution Buffer or sterile water (pH > 6.0) from the 65°C dry bath directly to the column matrix and incubate at room temperature for five minutes. Transfer the column to a 1.5 mL microcentrifuge tube and centrifuge at for 30 seconds to elute the DNA.
- Add another 20ul DNA Elution Buffer or sterile water (pH > 6.0) from the 65°C dry bath directly to the column matrix and incubate at room temperature for five minutes. Transfer the column to a new 1.5mL microcentrifuge tube and centrifuge at for 30 seconds to elute the DNA.
- You’re now ready to use this DNA.
- Quantify DNA from both final elution tubes with 1uL in the Superlab nanodrop (1st floor, east wing).
- As long as there is more than 30ng/uL, we are good.
This obviously needs to be cleaned up as well....
DNA extraction protocol for Streptococcus pneumoniae
- Spin down 1.2ml of growth in micro-centrifuge tubes (so, 2 sets of 625µl) at max speed for one minute. - Remove supernatant by shaking out liquid (no pipetting necessary). - Repeat these steps 4 times total in the same tube, to spin down all of the cells from all of the overnight growth. - Remove as much medium as possible with a pipet on the last round, without taking any cells. - Resuspend them in 1000 µl of 50mM Tris-10mM EDTA, pH 8 in a 2 ml eppendorf tube - Add 100 µl of 10 mg/ml lysozyme (absolute ± 1mg of lysozyme) - incubate at 37°C for 10 minutes - Add 5 µl 10 mg/ml proteinase K - incubate at 37°C for 30 minutes - Add 100 µl 10% SDS - Incubate at 37°C until lysis is complete (max 2h) = until suspension is clear - centrifuge for 5 min at 13.000 rpm - Transfer 900 µl supernatant to fresh 2 ml tube - Add 450 µl phenol and 450 µl SEVAG (or 900 µl 25:24:1 phenol/chloroform/isoamylalcohol mix), mix cautiously. - Centrifuge 5 min at 13.000 rpm and transfer 800 µl of the upper phase to a new 2ml tube - Add 400 µl phenol and 400 µl SEVAG (or 800 µl 25:24:1 phenol/chloroform/isoamylalcohol mix), mix cautiously. - Centrifuge 5 min at 13.000 rpm and transfer 700 µl of the upper phase to a new 2ml tube - Add 700 µl of SEVAG, mix cautiously - Centrifuge 5 min at 13.000 rpm and transfer 600 µl of the upper phase to a new 2ml tube - Add 1 ml cold (4°C) 100% EtOH at room temp. Mix by inverting the tube several times. - Leave at -20°C for 15 min. - Centrifuge for 5 min at max speed at room temp - remove all supernatant - Add 1 ml of cold (-20°C) 70% EtOH and invert the tube several times - centrifuge for 5 min at max speed at room temp - Remove all supernatant carefully - Dry at room temp (can use the vacuum) - Dissolve DNA in 100 µl MQ o/n
SEVAG: 24:1 chloroform:isoamylacohol
Streptococcus pneumoniae DNA extraction protocol
- Get strains from -80° freezer - Bring blood plate, inoculating loops, empty large test tube (as a container for dirty inoculating loops), and gloves to -80° freezer on 4th floor. - Wear gloves when streaking plates. - Streak out strains onto 1/4 of a blood plate (so, 4 strains per plate). - Put dirty inoculating loops in empty test tube; later, dispose of them upstairs in the lab. - Incubate plates overnight at 37°, 5% CO2
- Grow overnight cultures of strains - Do this as late in the day as possible. - Set up tubes with 5ml of CMT broth. - Inoculate from plates with a small amount of bacteria from the blood plate. 1/2 cm of a streak is plenty. - Incubate tubes overnight at 37°, 5% CO2
-Lyse cells - Spin down 1.2ml of growth in micro-centrifuge tubes (so, 2 sets of 625µl) at max speed for one minute. - Remove supernatant by shaking out liquid (no pipetting necessary). - Repeat these steps 4 times total in the same tube, to spin down all of the cells from all of the overnight growth. - Remove as much medium as possible with a pipet on the last round, without taking any cells. - Add 900µl lysis buffer. Resuspend completely using a vortex. - Trouble re-suspending cells? Let set for 10-15 minutes and try again. - Add 100µl lysozyme. Vortex to mix. Incubate in 37° water bath for 10 minutes. - Add 10µl protease K. Vortex to mix. Incubate in 37° water bath for 30 minutes. - Add 9µl RNAase A and 100µl of 10% SDS. Invert several times to mix. Incubate in 37° water bath until cultures are clear (minimum of 10 minutes, up to 1 hour). Invert occasionally to mix. - Spin down at max speed for 10 minutes. - Remove the top 1000µl (and only 1000µl) supernatant without getting the cell gunk at the bottom of the tube, and transfer to a new micro-centrifuge tube. If you disturb the cell gunk pellet, re-spin. Make sure no cell gunk is transferred! - Put tubes in Speed-Vac in the equipment room on high, with the tubes open. Stop when the samples are at approximately 200µl (approximately 90 minutes).
- Qiagen Blood and Tissue DNA extraction kit - Add 200µl 100%, 4° ethanol to tube. Invert gently, 20-30 times until you can see the DNA precipitate. If you cannot see a precipitate for most of your samples, stop. Lysis has failed. - Transfer whole volume of tube, including the DNA, to a spin column and collection tube. - Spin at 6000 g for 1 minute. - Discard collection tube; place column in new collection tube. - Add 500µl AW1. Spin at 6000g for 1 minute - Discard collection tube; place column in new collection tube. - Add 500µl AW2. Spin at 20,000g for 3 minutes. - Discard collection tube; place column in new micro-centrifuge tube. - Elute DNA with 200µl MQ water. Let water sit on filter for 10 minutes, in the 55° dry bath. - Spin down at 6000g for 1 minute. - Elute DNA with another 200µl MQ water into the same tube. Let water sit on filter for 10 minutes, in the 55° dry bath. - Spin down at 6000g for 1 minute.
-Quantify DNA - Quantify with 2µl using the Nanodrop on the 3rd floor. - Less than 15ng/µl? Extraction has failed. - Between 15ng/µl and 30ng/µl? Speed-Vac to get the volume from 400µl to 200µl. Then, redo Nanodrop measurement. - More than 30ng/µl? Excellent! Write concentration on the tube. Record concentration in lab notebook. - Store in -20° freezer.